OTUB1/ NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death

doi:10.21203/rs.3.rs-3421483/v1

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OTUB1/ NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death

https://doi.org/10.21203/rs.3.rs-3421483/v1

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published 23 Apr, 2024

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Pancreatic cancer is one of the most fatal cancers in the world. A growing number of studies have begun to demonstrate that mitochondria play a key role in tumorigenesis.Our previous study reveals that NDUFS2 (NADH: ubiquinone oxidoreductase core subunit S2), a core subunit of the mitochondrial respiratory chain complex I, is unregulated in Pancreatic adenocarcinoma (PAAD). However, its role in the development of PAAD remains unknown. Here, we showed that NDUFS2 played a critical role in the survival, proliferation and migration of pancreatic cancer cells by inhibiting mitochondrial cell death. Additionally, bioinformatics analysis in clinical samples indicated that the expression of NDUFS2 was positively correlated with a deubiquitinases, OTUB1. Overexpression of OTUB1 increased NDUFS2 expression at the protein level, while knockdown of OTUB1 restored the effects in vitro. Accordingly, overexpression and knockdown of OTUB1 phenocopied those of NDUFS2 in pancreatic cancer cells, respectively. Mechanically, NDUFS2 was deubiquitinated by OTUB1 via K48-linked polyubiquitin chains, resulted in an elevated protein stability of NDUFS2. Moreover, the growth of NDUFS2-overexpressed pancreatic cancer xenograft tumor was promoted in vivo, while the NDUFS2-silenced pancreatic cancer xenograft tumor was inhibited in vivo. In conclusion, we revealed that OTUB1 increased the stability of NDUFS2 in PAAD by deubiquitination, and this axis plays a pivotal role in pancreatic cancer genesis and development.

Health sciences/Biomarkers/Prognostic markers

Biological sciences/Cancer/Tumour biomarkers

Pancreatic cancer

OTUB1

NDUFS2

Deubiquitylation

PAAD is the fourth leading cause of all cancer-related deaths, with an overall 5-year survival rate of less than 10%, and it is expected to occupy the second place in 2030 [1, 2]. The poor prognosis of this disease is majorly attributed to the late diagnosis, frequent metastases, and limited treatment options [3]. Up to date, early diagnosis and prompt surgical intervention are the only effective means of improving the prognosis of those patients [4]. Unfortunately, even with a successful first-stage surgical resection, the prognosis is still poor, with a no more than 42% 2-year survival rate, due to the high recurrence rate [5]. However, the underlying mechanism is poorly understood.

Mitochondria play a crucial role in cellular bioenergetics and apoptosis, and thus are important to support cell function and in determination of cell death pathways [6]. The alterations in mitochondrial function and dynamics have been connected with metabolic diseases, neurological disorders and cancer progression as well [7]. NDUFS2, encoded by nuclear DNA, is localized in mitochondria and is one of the core subunits of the mitochondrial respiratory chain complex I. It has been shown that interfering with the intracellular NDUFS2 inhibited the activity of Complex I, reduced the intracellular ATP production, and thereby suppressed tumor growth and metastasis [8]. Elevated expression of NDUFS2 is associated with overall survival of several kinds of cancers, including lung, breast, and ovarian cancers [9]. It has been reported that the NDUFS2 is a prognostic biomarker of lung cancer and it has the potential to be widely used in future clinical settings [10]. Our previous study has demonstrated that NDUFS2 levels were elevated in pancreatic cancer tissues compared to adjacent normal tissues [11]. However, the role of NDUFS2 in pancreatic cancers remains uncovered.

The ubiquitin system is an enzymatic cascade that conjugant amino ubiquitin or polyubiquitin chains to target proteins, thereby promoting degradation or changing their activities [12]. Deubiquitinases (DUBs), a large class of proteases that cleaves mono- or polyubiquitin from target proteins, are considered to be a potential therapeutic target for various diseases [13, 14]. OTUB1, one of the protein deubiquitinases, is widely involved in various physiological and pathological processes and abundant expressed in many human tumors such as colorectal, lung, liver, ovarian and esophageal cancers [15–19], which is also strongly associated with the prognosis of those diseases. A recent report showed that OTUB1 could accelerate metastasis of pancreatic cancer by inhibiting FOXM1 degradation [20]. But its role in pancreatic cancers has not yet been fully demonstrated.

Considering the importance of OTUB1 and NDUFS2 in mitochondrial metabolism and tumorigenesis, we hypothesized that they may be involved in mitochondrial functions and tumorigenesis of pancreatic cancer. Thus, pancreatic cell lines and clinical surgical specimens were collected to verify our hypothesis. The primary aim of this study was to investigate whether OTUB1 or NDUFS2 or both of them can be used as new prognostic biomarkers and/or therapeutic targets for pancreatic cancer and shed new lights into exploring therapeutic methods for pancreatic cancer.

NDUFS2 is a promoter of cell growth, proliferation and migration in Pancreatic Cancer cells.

In our previous study, we identified NDUFS2 as a downstream factor of PTPMT1 in pancreatic cancer [11], but the precise role has not yet been validated. To determine the biological function of NDUFS2 in pancreatic cancer, gain- and loss- of function assays were performed with a pancreatic cancer cell line, Panc05.04. Overexpression of NDUFS2 significantly promoted the cell proliferation, whereas knockdown of it apparently inhibited the proliferation, revealed by CCK8 assay (Fig. 1A and 1B left panel). The knockdown and overexpression efficiencies were verified by Western blot (Fig. 1A and 1B right panel). Consistently, flow cytometric analysis showed that overexpression of NDUFS2 notably promoted the cell-cycle progression exhibited by elevated S-phase percentage (Fig. 1C, 1E), and it was impeded at G0/G1 phase in NDUFS2-silenced cells (Fig. 1D,1F). Additionally, colony formation capability of NDUFS2-overexpressed cells was magnified in contrast to the control (Fig. 1G), and it was dampened when NDUFS2 was knocked down compared to the control (Fig. 1H). Furthermore, wound healing and transwell migration assays showed that the mobility of NDUFS2-overexpressed cells was markedly increased, whereas that of NDUFS2-silenced cells was weakened (Fig. 1I-1L). Accordingly, the expression of E-cadherin was upregulated and Vimentin was downregulated compared to the control in NDUFS2-silenced cells (Fig. 1M). Those results indicated a critical role of NDUFS2 in pancreatic cancer cell growth, cell-cycle progression and migration.

NDUFS2 is an essential factor in mitochondrial membrane dynamics and ATP production in pancreatic cancer cells.

Given that NDUFS2 is a core subunit of the mitochondrial respiratory chain complex I, we inferred that it may regulate such cellular processes through modulating mitochondria. Therefore, mitochondrial membrane potential (MMP) was measured by JC-1 in NDUFS2-overexpressed and–silenced cells, respectively. Overexpression of NDUFS2 significantly increased the MMP in Panc05.04 cells, and thereby inhibited mitochondrial cell death (Fig. 2A,2C). Accordingly, knockdown of NDUFS2 markedly decreased the MMP, accompanied by aggravated mitochondrial cell death (Fig. 2B,2D). Similar results were observed in another pancreatic cancer cell line, Aspc-1 (Fig. 2E-2H). In addition, confocal analysis revealed that the mitochondria, stained by Mito Tracker, in the cytoplasm of NDUFS2-silenced cells were considerably shorter and fewer than those in the control cells (Fig. 2I). Those results suggested that NDUFS2 has a pivotal role in mitochondrial dynamics and cell fate determination in pancreatic cancer cells. Mitochondrial dynamics encompass processes of fusion and fission, with Drp1 participating in fission, while mitofusins(Mfn2) is associated with fusion[21].We also examined the expression of Drp1 and Mfn2 after knockdown of NDUFS2.The result showed that the expression of Drp1 was downregulated while Mfn2 was upregulated ,which means intracellular mitochondrial division decreased and mitochondrial fusion increased (Fig. 2J). This causes a decrease in the number of mitochondria in the cells.

To further validate the effects of NDUFS2 on cellular redox function and energy production, the NADPH/NADP⁺ ratio and ATP concentration in the culture media were measured. Overexpression of NDUFS2 increased the NADPH/NADP⁺ ratio and ATP concentration in Panc05.04 cells in contrast to the control (Fig. 2K and 2M), and knockdown of it decreased the NADPH/NADP⁺ ratio and ATP concentration in accordance (Fig. 2L and 2N), suggesting that NDUFS2 plays a key role in mitochondrial functions. Collectively, those data suggested that NDUFS2 plays an indispensable role in mitochondrial function and homeostasis in pancreatic cancer cells, which determined the cell fate.

OTUB1 and NDUFS2 are upregulated in pancreatic cancer

To find the potential regulators of NDUFS2, we screened the TCGA database and looked for candidates that could play a role in mitochondria, and OTUB1 was highlighted. Then we screened the expression of OTUB1 gene in major human cancer species using the TCGA database and found that the expression of OTUB1 was significantly elevated in PAAD compared to the adjacent normal tissues (Fig. 3A). We filtered all dysregulated genes in pancreatic cancer profiles from TCGA database, and identified OTUB1 and NDUFS2 were both upregulated in pancreatic cancer among all the upregulated genes, deciphered by the Volcano map (Fig. 3B). Meanwhile, we also found that the level of NDUFS2 was positively correlated with OTUB1 via GEPIA online database (R = 0.42; P < 0.01; Fig. 3C). Statistical analysis indicated that the expression of OTUB1 and NDUFS2 in pancreatic cancers was significantly higher than those in normal pancreatic tissues at the transcriptional level (Fig. 3D,3E). To further confirm these results, pancreatic cancer and adjacent tissue specimens were collected during clinical surgery, western blot assay was performed to measure the expression of OTUB1 and NDUFS2. The result showed that OTUB1, as well as NDUFS2, were abundantly expressed in pancreatic cancers in contrast to the adjacent tissues (Fig. 3F). Confocal assay showed that OTUB1 and NDUFS2 were co-localized and unregulated in pancreatic cancer tissues compared to the adjacent normal tissues (Fig. 3G). In vitro assays further indicated that overexpression of OTUB1 in pancreatic cancer cells increased the expression of NDUFS2(Fig. 3H,3I), and knockdown of OTUB1 decreased the expression of NDUFS2(Fig. 3J,3K). These results suggested that OTUB1 could be a co-regulator of NDUFS2 in pancreatic cancer progression.

OTUB1 mimicked the role of NDUFS2 in modulating cell growth, proliferation and migration in pancreatic cancer cells.

In the previous results, we identified OTUB1 as a NDUFS2 stabilizer in pancreatic cancer cells. Thus, we conjectured that the effects of OTUB1 on cell phenotype may be similar with NDUFS2. Overexpression and knockdown assays were performed in Panc05.04 cells to verify our assumption. The results showed that overexpression of OTUB1 significantly promoted cell proliferation, whereas knockdown of it apparently inhibited the proliferation, revealed by CCK8 assay (Fig. 4A and 4B left panel). The knockdown and overexpression efficiencies were verified by Western blot (Fig. 4A and 4B right panel). Consistently, flow cytometric analysis showed that overexpression of OTUB1 notably promoted the cell-cycle progression exhibited by elevated S-phase percentage (Fig. 4C,4E), and it was impeded at G0/G1 phase in OTUB1-scilenced cells (Fig. 4D,4F). Additionally, colony formation capability of OTUB1-overexpressed cells was magnified in contrast to the control (Fig. 4G), and it was dampened when OTUB1 was knocked down compared to the control (Fig. 4H). Furthermore, wound healing and transwell migration assays showed the mobility of OTUB1-overexpressed cells was markedly increased, whereas that of OTUB1-silenced cells was weakened (Fig. 4I-4L). Accordingly, the expression of E-cadherin was upregulated and Vimentin was downregulated compared to the control in OTUB1-silenced cells (Fig. 4M). Those results revealed broadly consistency between OTUB1 and NDUFS2 in the regulation of pancreatic cancer cell growth, cell-cycle progression and migration.

The similarities between OTUB1 and NDUFS2 in manipulating mitochondrial membrane dynamics and ATP production in pancreatic cancer cells.

Given that OTUB1 is a dominant modulator of protein stabilization of NDUFS2, which is a core subunit of Complex I, we inferred that it may modulate mitochondrial dynamics and functions in the same way as NDUFS2. Therefore, mitochondrial membrane potential (MMP) was measured by JC-1 in OTUB1-overexpressed and –silenced cells, respectively. Overexpression of OTUB1 significantly increased the MMP in Panc05.04 cells, and thereby inhibited mitochondrial cell death. In accordance, knockdown of OTUB1 markedly declined the MMP, accompanied by aggravated mitochondrial cell death (Fig. 5A and 5B). Statistical analysis indicated significant differences between the two groups (Fig. 5C and 5D). Similar results were observed in another pancreatic cancer cell line, Aspc-1 (Fig. 5E-5H). In addition, confocal analysis revealed that the mitochondria, traced by Mito Tracker staining, in the cytoplasm of OTUB1-silenced cells were considerably shorter and fewer than those in the control cells (Fig. 5I). Those results suggested that like NDUFS2, OTUB1 has a pivotal role in mitochondrial dynamics and cell fate determination in pancreatic cancer cells.

To further validate the function similarity of OTUB1 on cellular redox function and energy production, the NADPH/NADP⁺ ratio and ATP concentration in the culture media were determined. Overexpression of OTUB1 increased the NADPH/NADP⁺ ratio and ATP concentration in Panc05.04 cells in contrast to the control (Fig. 5J and 5L), and knockdown of it decreased the NADPH/NADP⁺ ratio and ATP concentration in accordance, suggesting a major role of OTUB1 in mitochondrial functions (Fig. 5K and 5M). Collectively, those data suggested that OTUB1 behaved much similarly with NDUFS2 in regulating mitochondrial membrane dynamics and ATP production in pancreatic cancer cells.

OTUB1 is a major regulator of NDUFS2 stability in pancreatic cancer.

Giving that the expression of OTUB1 and NDUFS2 exhibited tightly correlations in vitro, we presume that OTUB1 may stabilize NDUFS2 by direct interaction. Co-immunoprecipitation assay showed that endogenous OTUB1 and NDUFS2 interacted with each other in PANC05.04 cells (Fig. 6A and 6B). To examine whether the promoting role of OTUB1 in pancreatic cancer cells was mediated by NDUFS2, rescue experiments were performed in Panc05.04 cells. As shown in Fig. 6C, the expression of NUDFS2 decreased significantly after being transfected with sh-NDUFS2 plasmid, but these decreases were abolished after co-transfection with OE-OTUB1 plasmids. Meanwhile, the expression of NUDFS2 was overexpressed significantly after being transfected with OE-NDUFS2 plasmid, but the overexpression was abolished by co-transfection with sh-OTUB1 plasmids (Fig. 6D), suggesting that OTUB1 could promotes the expression of NDUFS2.

To further test that whether OTUB1could promote NDUFS2 protein degradation, MG132, chloroquine and CHX assay were used. We found that MG132, which is a proteasome inhibitor, could promote the expression of NDUFS2 at protein level in a time-dependent manner (Fig. 6E). Moreover, the expression of NDUFS2 was reduced when OTUB1 was knockdown, and MG132 could reverse this process (Fig. 6F). In contrast, there was no significant changes observed when treated with the lysosomal inhibitor Chloroquine, which means NDUFS2 is not degraded in the lysosomal pathway (Fig. 6G). Additionally, the expression of NDUFS2 was dramatically reduced after OTUB1 knockdown when treated with protein synthesis inhibitor, CHX (Fig. 6H). These above results suggesting that OTUB1 maybe involved in the ubiquitination degradation of NUDFS2.

The downregulation of NDUFS2 in OTUB1-deficient cells prompt us to test whether OTUB1 is a potential DUB for NDUFS2. Since ubiquitination of NDUFS2 has not been reported, we tested whether NDUFS2 undergoes ubiquitination modification. As shown in Fig. 6I, NDUFS2 could be notably ubiquitylated when co-transfected with exogenous ubiquitin. Meanwhile, Co-transfection with OTUB1 could significantly reduce the ubiquitination level of NDUFS2, suggesting OTUB1 may act as a DUB for NDUFS2 (Fig. 6J). As we known, the lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitination are the two most abundant types that contribute to the synthesis of poly-ubiquitin chains on protein substrates. Here, we focused on detecting changes in the K48- and K63-linked chains which may be involved in NDUFS2 degradation. We found that K48-linked ubiquitin completely abolished polyubiquitin chain formation on NDUFS2 (Fig. 6K), while K63-linked ubiquitin (K63-Ub) showed no obvious effect of polyubiquitin chain on NDUFS2, suggesting that OTUB1 promoted NDUFS2 degradation via K48-linked polyubiquitin chains. To further substantiate our conclusions, we checked the effect of OTUB1 on endogenous ubiquitination of NDUFS2 in pancreatic cancer cells. The results showed that overexpression of OTUB1 notably decreased the ubiquitination of NDUFS2, and knockdown of OTUB1 resulted in the increased the ubiquitination of NDUFS2 (Fig. 6L and 6M). Immunofluorescence assay (IF) suggested that NDUFS2 and OTUB1 were co-localized in the cytoplasm, and the expression of NDUFS2 was reduced when OTUB1 was knockdown (Fig. 6N). Taken together, those results indicated that OTUB1 may be directly participated in the proteasomal degradation of NDUFS2.

OTUB1 promotes the growth of Panc05.04 cells via increasing NDUFS2 in vivo

Previously, we characterized OTUB1 as a co-factor of NDUFS2 in the regulation of cell survival, proliferation and mobility in vitro and especially in clinical samples. To further verify the effects of OTUB1 on tumor formation in vivo, we injected Panc05.04 cells into subcutaneous tissues in nude mice after knockdown and overexpression of NDUFS2, respectively. When NDUFS2 was overexpressed, the growth of subcutaneous tumors was significantly increased compared to the control (Fig. 7A,7B,7E), and downregulation of NDUFS2 resulted in a significant decrease in the volume of subcutaneous tumors compared to the control (Fig. 7A,7B,7F). Statistical analysis suggested that the growing rate was apparently reduced when NDUFS2 was knockdown (Fig. 7C), while it was apparently increased after overexpression of NDUFS2 (Fig. 7D). Additionally, IHC staining suggested that the expression of OTUB1 was increased in the NDUFS2-overexpressed group (Fig. 7G), and it was reduced in the NDUFS2-silenced group (Fig. 7G). The schematic diagram that OTUB1 regulate the ubiquitination of NUDFS2 was shown in Fig. 7H.

Pancreatic cancer is the fourth leading causes of cancer death in the USA and leads to an estimated 227 000 deaths per year worldwide [22]. Surgical resection at present offers the only chance of cure, but only about 4% of patients will live 5 years after diagnosis[23]. Since pancreatic cancer responds poorly to most chemotherapeutic agents, thus there is an urgent need to find new therapeutic approaches and targets.

Mitochondria, the main sites of oxidative phosphorylation and ATP biosynthesis, provide the majority of the energy required by aerobic cells for maintaining their physiological activities [24, 25]. Targeting mitochondria to manipulate cell death holds tremendous therapeutic potential across different diseases, including various kinds of cancers[26]. In this study, we found that NDUFS2 plays a critical role in the proliferation, cell cycle precession, colony formation and migration of pancreatic cancer cells. It was further verified that NDUFS2 promoted pancreatic cancer cell migration by regulating the expression of E-cadherin and vimentin. NDUFS2 perhaps affecting mitochondrial dynamics and functions in pancreatic cancer cells by altering MMP. Recently, NDUFS2 has been identified as a redox-sensitive oxygen sensor[27]. We verified that interfering with NDUFS2 inhibited intracellular ATP synthesis and decreased the NADPH/NADP⁺ ratio significantly which means NDUFS2 could play an important role in the ATP synthesis and redox system. In addition, we confirmed that the levels of NDUFS2 and OTUB1 were positively correlated through Co-IP, both of which were abundantly expressed in pancreatic cancer.

OTUB1 is one of the most highly expressed DUBs in cells [28]. DUBs are considered as potential novel therapeutic targets for various diseases [13].OTUB1 is overexpressed in a wide variety of human tumors, such as colon cancer, gastric cancer, bladder and breast cancer [29–33]. However, the functions of OTUB1 in pancreatic cancer cells is rarely reported. In this report, we found that OTUB1 phenocopied the function of NDUFS2. Co-IP assay and western blot assays further revealed that OTUB1 increased the protein stability of NDUFS2 by deubiquitylation. Taking together, our present study revealed that OTUB1 played a crucial role in the development of pancreatic cancer, and NDUFS2, identified as a substrate of OTUB1, might be responsible for the observed phenotypes in the absence of OTUB1 in pancreatic cancer.

A recent report showed that OTUB1 may accelerate metastasis of pancreatic cancer by inhibiting FOXM1 degradation [20]. Here, we showed that NDUFS2 inactivation inhibited tumor growth in a mouse xenograft pancreatic cancer model, implicating a potential role of NDUFS2 in the development of pancreatic cancer in vivo. Treatment targeting NDUFS2 may provide us novel approaches for the treatment of pancreatic cancer. Due to the widespread distribution and high cellular expression of OTUB1 and NDUFS2 in tissues, it may be difficult to inhibit their expression in clinic, but treatment targeting them may provide significant benefit to patients diagnosed with multiple tumors[34].

In summary, our study provides new insights into the biological function of OTUB1 and NDUFS2, indicating that targeted therapy against OTUB1 and/ or NDUFS2 could hold the premise to provide a new therapeutic method for the treatment of pancreatic cancer, but the specific mechanism by which the OTUB1/NDUFS2 axis regulates the mitochondrial death of pancreatic cancer cells needs to be further experimentally investigated and consolidated.

Cell lines and culture

The human pancreatic cancer ASPC-1 cell was cultured in RPMI-1640 (31870082; Gibco) supplemented with 10% FBS (16140071; Gibco) and 100X Penicillin-Streptomycin Solution(SV30010,hyclone), PANC05.04 cells were cultured in F12k media (#21127022, Gibco) supplemented with 10% FBS and 100X Penicillin-Streptomycin Solution. Cells were incubated in a humidified incubator at 37°C with 5% CO₂.

Cell Transfection

sh-OTUB1, OE-OTUB1, sh-NDUFS2, OE-NDUFS2 and their each control plasmids and lentivirus were purchased from Genechem (Shanghai, China) company. Transient transfections were performed with jetPRIME (#150 − 15, Polyplus, France) transfection reagent following the manufacturer’s instructions. The sequence of sh-NDUFS2-1 was 5’-GCAGATGTCGTTGCCATCATA-3’; the sequence of sh-NDUFS2-2 was 5’GCTGTTATGTACCCAAGCAAA-3’. The sequence of sh-OTUB1-1 was 5’-GCAAGUUCUUCGAGCACUU-3’; the sequence of sh-OTUB1-2 was 5’-GCGACUCCGAAGGUGUUAATT’.The overexpression and knockdown cell lines were generated by lentivirus transduction.

Cell proliferation assay

Cell viability was quantified using the Cell Counting Kit-8 assay(B34304, Selleckchem). Cells were seeded into 96-well plates at a density of 3x10³ cells/well and were cultured in a humidified atmosphere with 5% CO₂ at 37°C. Subsequently, 10 µl CCK-8 reagent was added to each well. The absorbance was measured at 450 nm after 2h. The CCK-8 assay was performed every 24 hours for 4 consecutive days.

Colony formation assay

For colony formation analysis, 1,000–1,500 cells per well were plated in six-well plates and allowed to grow for 14 days after the indicated treatments, then we fixed the cells with 4% paraformaldehyde(P1110, Solarbio), and stained the cells with 5% crystal violet (G1063,Solarbio).

Cell Cycle Assay

For the cell cycle assay, the Cell Cycle Staining Kit (CCS012, Multi Sciences Biotech Co., Ltd.) was used according to the manufacturer's protocol. Briefly, cells were harvested and fixed in 70% ethanol at 4°C overnight. Cells were then treated with 1ml DNA staining solution and 10µl permeabilization at 37°C in the dark for 30 mins. Cells were then analyzed via flow cytometry.

Transwell Migration Assay

2 × 10⁵ cells which were suspended in 1.5ml serum free culture media were added to the top chamber of 6-well transwell plates (3428, Corning), and 2.6ml culture media containing 10% FBS was added to the bottom chamber. After incubating at 37℃ for 48 h, the chambers were washed with PBS twice, and these cells which migrated to the bottom chambers were fixed with paraformaldehyde and stained with crystal violet. Then the number of transitional cells in all chambers was calculated in the 5 visual fields.

Wound Healing Assay

Cells were seeded on 6-well plate and grew to the pavement overnight. After 24 hours of transfection, a channel was drawn on the monolayer cells with 10µl micropipette tip. Then cells were washed with PBS twice and cultured in serum-free culture media at 5% CO₂, 37℃ for an additional 24 h. Photographs were taken by an inverted Leica phase contrast microscope at 0 h and 24 h.

Measurement of Mitochondrial membrane potential.

To measure mitochondrial membrane potential (MMP), cells were washed with PBS and incubated with JC-1 (C2005, Beyotime Biotechnology) at 37°C for 20 min in the dark. After incubation with the dye, the plates were washed three times with PBS. Fluorescence was measured first at excitation/emission 485/580 nm (red) and then at excitation/emission 485/530 nm (green) using a flow cytometer (BD, Fortessa). The results were analyzed with FlowJo software.

Analysis of mitochondrial activity

Cells were plated on poly-l-lysine-coated glass coverslips. Afterward, we stained the cells with 100 nM Mito-Tracker(C1048, Beyotime Biotechnology)Deep Red/Green staining solution in the dark for 30 min to visualize mitochondria, followed by confocal imaging (ZEISS, Germany).

NADP⁺/NADPH and ATP Assay

For measurements of NADP⁺/NADPH ratio and ATP concentration, cells were harvested and treated with appropriate buffers as indicated by the providers. In particular, NADP+/NADPH ratios were determined using a NADP⁺/NADPH Assay kit (S0179, Beyotime) and were detected using a colorimetric assay under a microplate reader (Feyond-A400, ALLSHENG, China) with detection wavelengths of 570 nm, respectively. The ATP concentration was calculated using an enhanced ATP assay kit (S0027, Beyotime) by measuring chemiluminescence with a luminometer plate reader.

Western blotting

Cells were harvested 48h after transfection of the plasmid for immunoblotting analysis.Cells were lysed using RIPA buffer (P0013B, Beyotime Biotechnology). Subsequently, total protein was separated using SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked and were subsequently incubated at 4°C overnight with the following primary antibodies against: GAPDH (2118, 1:1000; CST), OTUB1 (270959, 1/1000; Abcam),beta-Tubulin (16305, 1:2000; CST), NDUFS2 (sc-390596, 1:1000; MDL Biological, Inc.), E-cadherin(ab233611, 1/10000; Abcam),Vimentin (ab92547, 1/2000; Abcam); Drp1 (Cell Signaling, 1:1000, 8570S); Mfn2 (Cell Signaling, 1:1000,11925S);HA-Ub(CST,3936S); HA-K48-Ub(CST,8081S); HA-K63-Ub(CST,5261S). All antibodies were used according to the manufacturer's protocol. Following primary incubation, membranes were incubated with secondary antibodies, HRP-conjugated goat anti-rabbit IgG (PR30011, 1:20000; Proteintech Co., Ltd.) and HRP-conjugated goat anti-mouse IgG (PR30012, 1:20000; Proteintech Co., Ltd.) at room temperature for 1 h. Proteins were visualized using ECL Reagent (42029053, Millipore Sigma).

Drugs and inhibitors

10µM MG132(HY-13259, MCE) which is a potent proteasome inhibitor and 200µM Chloroquine (HY-17589A, MCE) which is a lysosomal enzyme inhibitor, was used to treat pancreatic cell lines for 8h; 20 µg/ml CHX (HY-12320, MCE), which is an inhibitor of protein synthesis, was used to inhibit the protein synthesis at different time.

Immunofluorescence staining and co-immunoprecipitation

Slides containing human pancreatic tumor tissues and adjacent normal pancreatic tissues were used in our experiment. The slides were incubated with primary antibodies at 4°C overnight. The following day, after incubation with the corresponding secondary antibody, the nuclei were stained with DAPI (Yeason,40728ES03) before observing by confocal laser microscopy. Co-immunoprecipitation analysis was carried out using the Pierce Co-Immunoprecipitation Kit (88804; Thermo Fisher) according to the manufacturer’s instructions. primary antibodies against: Ub (3936s, 1:1000; CST), IgG light chain (SA00001-7L; 1:1000; Proteintech, Inc.),Flag(ABT2010,1:1000,Abbkine) were used according to the manufacturer's protocol.

Immunochemical (IHC)-staining

IHC analyses were done by the Mango Bioservice company (Beijing, China) using primary antibodies against OTUB1, and NDUFS2 at 1:100. The expression levels of OTUB1 and NDUFS2 were assessed using the H score approach as described previously[11].

Patients and tissue handling

Between September 2022 and June 2023, 8 pairs of PAAD tissues along with neighboring non-tumorous pancreatic tissues were acquired from the Department of Hepatobiliary surgery of PLA general hospital during surgery and we obtained informed consent from each subject. All samples were preserved in 4% paraformaldehyde solution waiting for the next experiment.

Animal Studies

Panc05.04 cell lines was used for xenograft tumor model. Five-week-old male nude mice (SPF Bio-Technology Co. Ltd, Beijing) were injected subcutaneously with 1 × 10⁷ cells with sh-control, sh-OTUB1-1, sh-OTUB1-2 and OE-OTUB1 groups (3 nude mice in each group) suspended in 100µl PBS and mixed with 100µl Matrigel (356231, BD Biosciences, America) and PBS. These cells were implanted subcutaneously into the dorsal flank sides of the mice. Once the diameter of tumors reached nearly 2 mm, the volume of tumor was measured daily for 2 weeks. The lengths and widths of the tumors were measured every 3 days. Tumor volume (mm³) was calculated as length × (width)²×0.52. On the 15th day, these mice were killed and tumors were extracted and measured. The tumors were fixated with paraformaldehyde, then immunohistochemistry (IHC) staining was performed for OTUB1, NDUFS2, and HE. All procedures involving mice were approved by the University Committee on Use and Care of Animals at the Capital Medical University and met all regulatory standards.

Bioinformatics data

Differential expression data of OTUB1 and all the dysregulated genes in PAAD were obtained from TCGA (https://portal.gdc.cancer.gov/) database. The transcriptional expression of OTUB1 and NDUFS2 were extracted from Gepia2(http://gepia.cancer-pku.cn/) database.

Statistical Analysis

Data analyses were carried out using SPSS 21.0 (IBM Corp., USA) as well as GraphPad Prism 5 (GraphPad Software, CA). All results were from three independent experiments. Normally distributed data were given as mean ± SEM. Statistical comparisons between groups were assessed using the Student’s T-test (two-tailed) and ANOVA analysis. Statistical significance was denoted by P < 0.05(* ,p < 0.05;**,P < 0.01;***,p < 0.001).

Acknowledgments

This research work was supported by Beijing Post-doctoral Foundation (Grant No.2023-ZZ041)

Conflict of Interest Statement

The authors declare no competing interests.

Author Contribution Statement

Fengjun Xiao designed this study. Yajin Liao and DongDong Lin supervised the research. Xiaodong Huang conducted the main experiments. Li-Du, Xiao-Chen Cheng, Yu-Xin Lu, Qiao-Wei Liu contributed to technical Support. Yi-Wu Wang helped to analyze data. Xiaodong Huang wrote the manuscript. All authors read and approved the final manuscript.

Data Availability Statement

The published article includes all data sets generated/analyzed for this study.

Ethics Statement

All aspects of this study were approved by Institutional Research Ethics Committee of

Xuanwu Hospital Medical University.

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You are reading this latest preprint version

OTUB1/ NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

OTUB1 and NDUFS2 are upregulated in pancreatic cancer

OTUB1 promotes the growth of Panc05.04 cells via increasing NDUFS2 in vivo

Discussion

Material and Methods

Cell lines and culture

Cell Transfection

Cell proliferation assay

Colony formation assay

Cell Cycle Assay

Transwell Migration Assay

Wound Healing Assay

Analysis of mitochondrial activity

NADP⁺/NADPH and ATP Assay

Western blotting

Drugs and inhibitors

Immunofluorescence staining and co-immunoprecipitation

Immunochemical (IHC)-staining

Patients and tissue handling

Animal Studies

Bioinformatics data

Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

OTUB1/ NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

OTUB1 and NDUFS2 are upregulated in pancreatic cancer

OTUB1 promotes the growth of Panc05.04 cells via increasing NDUFS2 in vivo

Discussion

Material and Methods

Cell lines and culture

Cell Transfection

Cell proliferation assay

Colony formation assay

Cell Cycle Assay

Transwell Migration Assay

Wound Healing Assay

Analysis of mitochondrial activity

NADP+/NADPH and ATP Assay

Western blotting

Drugs and inhibitors

Immunofluorescence staining and co-immunoprecipitation

Immunochemical (IHC)-staining

Patients and tissue handling

Animal Studies

Bioinformatics data

Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

NADP⁺/NADPH and ATP Assay