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. 2024 Sep 11;50:102118. doi: 10.1016/j.tranon.2024.102118

MG53 suppresses tumor growth via transcriptional inhibition of KIF11 in pancreatic cancer

Xiao-Liang Wang a,b, Xiangfei He b, Tong Gao a, Xinyu Zhou a,b, Zobeida Cruz-Monserrate c, Allan Tsung a,b, Jianjie Ma a,b,⁎, Chuanxi Cai a,b,⁎
PMCID: PMC11416540  PMID: 39265509

Highlights

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    Pancreatic tumors grew more aggressively in MG53 knockout mice compared to wild type mice in animal models.

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    MG53 localizes to the nucleus of pancreatic cancer cells, exerting inhibitory effects on colony formation and proliferation.

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    Decreased MG53 and increased KIF11 expression were found in pancreatic cancer.

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    MG53 can bind to KIF11 promoter to transcriptionally suppress KIF11 expression, leading to cell cycle arrest in pancreatic cancer cells.

Keywords: TRIM72, Nuclear translocation, Cell proliferation, Syngeneic orthotopic transplantation

Abstract

Pancreatic ductal adenocarcinoma (PDAC) poses a formidable challenge in oncology due to its limited treatment options and poor long-term survival rates. Our previous work identified MG53, a member of the tripartite motif family protein (TRIM72), as a key player in tissue repair with potential applications in regenerative medicine. Despite the focus on MG53’s cytosolic functions, its nuclear role in suppressing pancreatic cancer remains unknown. Through orthotopic and subcutaneous transplantation studies in mice, we observed enhanced tumor growth in MG53-deficient mice compared to wild-type counterparts. The overexpression of KIF11, a motor protein crucial for cell mitosis regulation, has been linked to the aggressive proliferation of pancreatic cancer cells. Confocal imaging confirmed MG53′s presence in the nucleus of human pancreatic cancer cells, while functional assays demonstrated its impact on KIF11 expression and subsequent cell proliferation. Mechanistically, we revealed MG53′s transcriptional control over KIF11, leading to cell cycle arrest. Our findings position MG53 as a promising tumor suppressor in PDAC, offering a novel avenue for therapeutic intervention by regulating KIF11 expression.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) represents a significant challenge in oncology due to its high fatality rates, with a 5-year survival rate of <10 % [1]. Its aggressive nature and propensity for metastasis to distant organs underscores the urgency for effective treatment options. While surgical and systematic therapies offer some benefits, more effective treatments are needed to improve the poor outcomes in patients with PDAC [2]. The lack of targeted interventions highlights the need for in-depth exploration of PDAC's underlying susceptibility mechanisms and susceptibility factors.

Our research group has previously identified MG53, a member of the tripartite motif family protein also named as TRIM72, as a significant contributor to cell membrane repair [3]. Over the last 15 years, extensive studies from us and other investigators have investigated the biology of MG53 and its potential applications in regenerative medicine [[3], [4], [5], [6], [7], [8]]. Utilizing a genome-wide CRISPR gene-silencing approach, Chen et al. demonstrated that the knockout of TRIM72/MG53 leads to heightened tumor growth and metastasis in lung cancer [9], suggesting a potential tumor suppressor role for MG53. Reduced levels of MG53 in the bloodstream have been associated with an elevated risk of cancer development in human patients [10]. Our recent data support the notion that MG53 functions as a tumor suppressor by targeting G3BP2 or stress granule activity in non-small cell lung cancer [11].

KIF11, a member of the kinesin superfamily, plays a crucial role in the spindle formation during mitosis [12], and has been implicated in the proliferation of pancreatic cancer cells [13,14]. Targeting the hyperactivity of KIF11 through molecular approaches presents a promising avenue for PDAC therapy.

In the present study, we used a preclinical mouse model to orthotopically transplant murine pancreatic cancer cells [[15], [16], [17]] into both wild-type (WT) and MG53-knockout (KO) mice, and investigate whether MG53 plays a role in pancreatic tumorigenesis and examine how MG53 regulate the pancreatic cancer cell proliferation through its nuclear activity, specifically by transcriptionally controlling the expression of the KIF11 gene.

Methods

Ethics statement

All experimental procedures were conducted in adherence to the regulatory standards and ethical principles governing animal research, as stipulated by the Institutional Animal Care and Use Committee at the University of Virginia and/or The Ohio State University. Human PDAC biopsy samples were obtained through The Ohio State University Wexner Medical Center Total Cancer Care Protocol with an approved data use agreement.

Animal model of syngeneic orthotopic transplantation of murine pancreatic cancer cells

We established an animal model involving syngeneic orthotopic transplantation of murine pancreatic cancer cells. This model utilized MG53 knockout mice [3] and their wild-type littermates, all with a C57BL/6 background (RRID:IMSR_JAX:000664). The mice were injected with cells derived from pancreatic tumors of a genetically engineered mouse model (GEMM) designated LSL-KRasG12D/LSL-Trp53−/−/PDX1-CRE (KPC), which were transfected with enhanced firefly luciferase (KPC-Luc) [16,18,19]. The KPC-Luc cells were prepared in a single-cell suspension and mixed with HBSS and 20 % Matrigel (BD Biosciences). Subsequently, a suspension containing 1 × 105 KPC-Luc cells was meticulously implanted following established protocols [16,19]. Tumor growth was monitored by administering d-Luciferin (150 mg/mouse; LifeGold Biotechnolgy) and utilizing the IVIS Lumina II in vivo imaging system (PerkinElmer Inc.) located in the Small Animal Imaging Core at The Ohio State University. Imaging was conducted weekly until euthanasia became necessary, and bioluminescence was quantified using Living Image software (RRID:SCR_014247).

Subcutaneous transplantation of pancreatic cancer cells

KPC-Luc cells were also subcutaneously implanted into the flank region of each mouse, including MG53-knockout (KO) and wild-type (WT) littermates with a C57BL/J background, aged 8–10 weeks. Surgical procedures were conducted under anesthesia induced by inhalation of vaporized isoflurane. Each mouse received a subcutaneous injection of 5 × 105 KPC-Luc cells suspended in HBSS and 20 % Matrigel (BD Biosciences). Sacrifice was performed at week 3 post-surgery. Tumor dimensions were assessed weekly by measuring the length (L) and width (W) with a caliper, and tumor volume was calculated using the formula: tumor volume = [L × W2]/2.28). Following euthanasia, tumor masses were retrieved, and their weights were measured using a top-loading digital balance.

Cell culture

Pancreatic cancer cell lines and HEK293T cells were maintained under standard cell culture conditions at 37 °C with 5 % CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4.5 g/L glucose, l-glutamine, 100 units/mL penicillin and 100 g/mL streptomycin (Gibco, Grand Island, NY, USA), plus 10 % (v/v) fetal bovine serum (FBS, Thermo Scientific). Prior to experimentation, all cell lines were rigorously screened for the absence of mycoplasma contamination. Specifically, PANC-1 and MPANC96 cell lines were procured from the American Type Culture Collection (ATCC). Human Pancreatic Stellate Cells were obtained from ScienCell Research Laboratories (Catalog No 3830, Carlsbad, CA) and cultured in Stellate Cell Medium, following established protocol [20]. To express myc-tagged KIF11, the p-Receiver-Lv107-KIF11 plasmid was purchased from GeneCopoeia (EX-Z3227-Lv107). HEK293T cells were co-transfected with pCMS-MG53 [21] and p-Receiver-Lv107-KIF11 plasmids for 36 h. Following transfection, the cells were treated with or without 10 µM MG132, a proteasome inhibitor, for 8 h. Subsequently, the cells were collected and subjected to Western blot analysis to determine the protein levels of MG53 and KIF11.

RNA isolation and quantitative real-time PCR

Real-time quantitative polymerase chain reaction (qPCR) was performed to assess the gene expression levels of MG53 and EG5/KIF11 in cultured cells. Total RNA extraction from each sample was conducted using the Rneasy Fibrous Tissue Kit following the manufacturer's protocol (Qiagen#74,704). Subsequently, reverse transcription of 1 µg of RNA into complementary DNA (cDNA) was performed using the Bio-Rad iScript cDNA Synthesis Kit (Bio-Rad Cat#170–8891). The primer sequences utilized were as follows: MG53 forward primer: 5′-GTAAGGAGAAGAGTGTGGCTG-3′, MG53 reverse primer: 5′-AGGAACACCCGCATCTTG-3′; KIF11 forward primer: 5′-GCCGTTCTGGAGCTGTTGAT-3′, KIF11 reverse primer: 5′-CGCCCTCCAAGAGAATCCTG-3′. Subsequently, samples for real-time qPCR analysis were prepared according to the manufacturer's instructions using the iTaq Universal SYBR Green Supermix (Bio-Rad Cat#172–5124) and analyzed on a Bio-Rad CFX384 Real-Time system.

Subcellular fractionation

Equal numbers of human MPan96 and PANC1 cells were infected with either Ad-MG53 or a control adenovirus and harvested after 48 h of doxycycline induction for subcellular fractionation. This was accomplished using the Compartmental Protein Extraction Kit (Catalog K3013010, BioChain, CA, USA), following the manufacturer's instructions. In brief, cell samples were mechanically homogenized in buffer C, after which cellular compartments were sequentially separated using buffers C and N. The resulting cytosolic and nuclear fractions were then subjected to Western blot analysis.

Immunoblotting

Crude extracts obtained from either cells or pancreatic tumors were subjected to two washes with ice-cold phosphate-buffered saline (PBS) and subsequently lysed in RIPA buffer (composed of 10 mM Tris–HCl, pH 7.2, 150 mM NaCl, 1 % NP-40, 0.5 % SDS, and 0.5 % deoxycholate). The lysis buffer was supplemented with a cocktail of protease inhibitors (Sigma) and phosphatase inhibitors (Thermo Scientific). Following lysis, cell or tissue lysates were subjected to separation by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were washed with Tris-buffered saline Tween-20 (TBST), blocked with 5 % milk in TBST for 1 h, and then incubated with a custom-made monoclonal anti-MG53 antibody (clone 914) [4,22]. Immunoblots were visualized using an ECL Plus kit (Pierce). Additionally, antibodies against GAPDH (CST-2118, Cell Signaling), CCNB1 (CST-4138), Cdc2 (CST-77,055), Phospho-cdc2 (Tyr15) (10A11) (CST-4539), KIF11 (PA582394), and DOT1L (MA5–35,166), were used in this study. Multiple samples derived from different animals were routinely employed in each Western blot analysis. To depict the variability in band intensity among wild-type samples, band intensities were normalized to a control sample (typically one with intermediate intensity) and plotted as relative intensities in a scatter plot. Uncropped Western blotting images were submitted as a supplemental file.

Immunostaining and confocal imaging

Paraffin-embedded tumor sections, each measuring 5 µm in thickness, were subjected to de-paraffinization using xylene followed by gradual rehydration in 100 %, 95 %, 70 % and 50 % ethanol solutions. Antigen retrieval was then carried out. Immunohistochemical staining was conducted, and confocal images were captured using a Zeiss confocal laser-scanning microscope (Zeiss LSM780). Quantitative analysis of the images was performed using ImageJ (RRID:SCR_003070). For each group, a minimum of three sections from different subjects were examined.

Chromatin immunoprecipitation (ChIP) assay

After infection with either Ad-MG53 or Ad-Ctrl adenovirus, human PANC1 cells (4 × 106) were prepared for the ChIP assay using the Pierce Magnetic ChIP Kit (Catalog No 26,157, Pierce Biotechnology, IL, USA) following the manufacturer's protocol. The MG53 antibody (clone 914) was utilized to immune-precipitate DNA fragments, which were subsequently subjected to PCR amplification targeting the binding region of the KIF11 promoter. Normal rabbit IgG was employed as a negative control. The primers utilized in the ChIP assay were as follows: KIF11-P1-F: 5′-CACACACACACACACACAAAG-3′ (sense); KIF11-P1-R: 5′-GCAGTAGAAGCACAGAAGAGAG-3′ (antisense).

Luciferase reporter assay

A 908-base pair fragment of the KIF11 promoter was cloned and inserted into the pGL3-basic luciferase reporter vector (RRID:Addgene_11,994), resulting in the creation of the KIF11 promoter luciferase reporter plasmid. For the assay, HEK293T cells (RRID:CVCL_0063) were seeded and subsequently transfected with the designated promoter reporter plasmid (500 ng) along with the pRL-SV40 plasmid (20 ng). After 48 h of transfection, cellular lysis was performed using the Dual-Glo® Luciferase Assay Kit (Promega, WI, USA) following the manufacturer's protocol, and luciferase activities were quantified using the GloMax® 96 Microplate Illuminometer (Promega, WI, USA).

Cell cycle analysis

Cell cycle analysis was conducted using flow cytometry. PANC-1 human pancreatic cancer cells were infected with either Ad-MG53 or Ad-Ctrl for a duration of 12 h, followed by incubation with doxycycline (1 µg/ml) to induce MG53 expression for 48 h. Subsequently, after 48 h of doxycycline induction, cells were collected, washed twice with PBS, and fixed in ice-cold 70 % ethanol overnight at 4 °C. The following day, all samples underwent centrifugation at 3000 rpm for 4 min. The cells were then stained with propidium iodide (5 mg/ml), supplemented with RNase A, and analyzed using flow cytometry with Guava EasyCyte System (EMD Millipore Corporation, Burlington, MA, USA) [23]. DNA content was assessed under a blue laser (488 nm), and the maxima were recorded at the 615/20 nm filter. Data analysis was performed using FlowJo (RRID:SCR_008520) (BD Biosciences, Ashland, OR, USA).

Clonogenic assay

Before cell seeding, a 6-well plate was coated with 0.2 % gelatin (Sigma Cat# G9391) and incubated at 37 °C for 1 hour. PANC-1 cells were first infected with adenovirus expressing MG53 (Ad-MG53) or a control vector (Ad-Ctrl) for 12 h without doxycycline induction. Subsequently, two groups of 2000 suspended PANC-1 cells were transferred into each well of the 6-well plate. The culture medium containing 1 µg/ml doxycycline was refreshed every 2 days over a period of 10 days to facilitate colony formation. After incubation, the colonies were fixed with 4 % formaldehyde for 10 min and stained with 0.01 % (w/v) crystal violet in ddH2O for 30 min. Excess crystal violet was rinsed off with ddH2O, and the dishes were allowed to air dry before imaging analysis. The images were scanned using the Leica DMi8 microscope, and colonies larger than 5000 µm² were counted using ImageJ software.

Statistical analysis

Statistical analysis was conducted utilizing the Prism 9 software program (GraphPad Prism, RRID:SCR_002798). Data are presented as the mean ± standard error of the mean (SEM). Unpaired student's t-test or one-way analysis of variance (ANOVA) were employed for comparisons among different groups. Statistical significance was defined as p < 0.05.

Results

Aggressive pancreatic tumor growth in MG53-KO mice

We previously demonstrated the tumor suppressor role of MG53 by showing that MG53-KO mice have a higher propensity for developing lung tumors as they age [11]. Here we adopted a preclinical mouse model for syngeneic orthotopic transplantation of KPC-Luc cells (KPCs, 1 × 105) into the pancreas of WT and MG53-KO mice. KPCs are derived from pancreatic tumors in KRasG12D/Trp53-/-/Pdx1-Cre (KPC) mice [15,16] and have been transfected with enhanced firefly luciferase, facilitating in vivo bioluminescence imaging using the IVIS system for real-time quantification of pancreatic tumor growth. Initial observations showed similar luminescence levels at the implantation site in both WT and MG53-KO mice 1 day after orthotopic transplantation of KPCs (Fig. 1a, upper). However, quantitative analysis of bioluminescence at 28 days post-tumor cell implantation revealed that MG53-KO animals developed larger tumors compared to WT mice (Fig. 1b). At the endpoint, animals were sacrificed for tumor collection, and tumors harvested from MG53-KO mice exhibited significantly greater weights compared to those from WT mice (Fig. 1c-d). Collectively, these findings suggest accelerated pancreatic tumor growth in the absence of MG53.

Fig. 1.

Fig. 1

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Aggressive pancreatic tumor growth was observed in MG53-KO mice compared to WT mice three weeks after orthotopic transplantation of KPC-Luc cells into their pancreas. a-b. We developed a protocol for syngeneic orthotopic transplantation of KPC-Luc cells (1 × 105) into WT and MG53-KO mice, expressing enhanced firefly luciferase for in vivo bioluminescence imaging using the IVIS system to monitor pancreatic tumor growth dynamically. Initial luminescence levels at the implantation site were similar between WT and MG53-KO mice, but quantitative analysis revealed larger tumors in MG53-KO mice compared to WT mice 28 days post-implantation, indicating accelerated pancreatic tumor growth in the absence of MG53. c-d. At the study's end, animals were euthanized, and tumor collection revealed significantly heavier tumors in MG53-KO mice compared to WT mice, as demonstrated in the slide. Data are mean ± SEM; p value was calculated by unpaired Student's t-test and presented in the individual panels.

To further elucidate the anti-tumor role of MG53, we conducted subcutaneous transplantation of KPC-Luc cells (5 × 105) into both WT and MG53-KO mice, measuring volume at various points post-transplantation. Similar to the orthotopic transplantation, the rate of tumor volume increase was notably accelerated in MG53-KO mice compared to the WT control group (Fig. 2a). Upon completion of the experiment, animals were euthanized, and individual tumors from various animals were collected and imaged (Fig. 2b), revealing a significantly greater tumor weight in the MG53-KO group (Fig. 2b and c). Immunostaining of Ki67, a marker for cell proliferation, further demonstrated a significantly higher number of Ki67-positive cells in tumors collected from MG53-KO mice compared to WT (Fig. 2d), indicating enhanced cell proliferation capacity in KPCs in the absence of MG53. Overall, findings from both orthotopic and subcutaneous transplantation models of KPCs underscore the promotion of pancreatic tumor growth in MG53 deficient mice, highlighting MG53’s anti-tumor function.

Fig. 2.

Fig. 2

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Progressive tumor growth was observed in MG53KO mice compared to that in WT mice after subcutaneous xenograft of KPC cells. a. Faster tumor growth was noted in MG53 KO mice compared to WT mice over three weeks post subcutaneous xenografts of KPC cells (5 × 105), as measured by tumor volume. b. At the end of three weeks, tumors collected from MG53 KO mice were notably larger and heavier compared to those from WT mice. c. Quantitative analysis for the tumor weight shown in panel b. d. To evaluate cancer cell proliferation among groups, immunostaining of Ki67, a cell proliferation marker, was conducted on tumor sections, revealing a notably higher number of Ki67-positive cells in tumors from MG53-KO mice compared to tumors from WT mice. Data are mean ± SEM; p value was calculated by unpaired Student's t-test and presented in the individual panels.

Overexpression of MG53 suppresses the proliferation and colony formation PANC-1 cells

To assess the therapeutic potential of MG53, we developed a viral-based gene delivery system aimed at modulating MG53 expression in cancer cells through doxycycline induction [11]. The TRE-tPA-MG53 plasmid was incorporated into adenovirus (Ad-MG53) for efficient transduction of PANC-1 cells. Live cell imaging revealed that doxycycline-induced MG53 expression (see Fig. 5a) inhibited the proliferation of PANC-1 cells compared to those infected with the control adenovirus (Ad-Ctrl) at 48 h post-infection (Fig. 3a). This observation was corroborated by quantitative analysis based on three independent cell counts conducted at 48 and 72 h following MG53 overexpression in PANC-1 cells (Fig. 3b). A colony formation assay was conducted by seeding 2000 PANC-1 cells infected with either Ad-Ctrl or Ad-MG53 into a 6-well plate and culturing them for 10 days in the presence of doxycycline. The surviving colonies were fixed, stained with crystal violet for visualization, and quantified to assess plating efficiency. As shown in Fig. 3c, the number of colonies in cells with inducible MG53 expression was significantly lower compared to the control group. Additionally, the size of colonies in the group with inducible MG53 expression was noticeably smaller than those in the group without MG53 overexpression (Supplemental Fig. S1). These findings collectively demonstrate that overexpression of MG53 effectively suppresses the proliferation of human pancreatic cancer cells.

Fig. 5.

Fig. 5

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MG53 transcriptionally regulates the gene expression of MG53. a. An adenoviral-based gene delivery system was created to control MG53 expression in cancer cells using doxycycline induction, resulting in significantly increased MG53 expression and decreased KIF11 expression in PANC-1 cells upon treatment with 1 µg/ml Dox. b. Western blot analysis of tumors from WT and MG53-KO mice, along with normal pancreas and cultured KPC cells, revealed high KIF11 expression in KPCs but not in normal WT pancreas, significantly lower KIF11 levels in tumors from WT mice compared to tumors from MG53-KO mice, and significant level of MG53 protein in tumors from WT mice but not detectable in tumors from MG53-KO mice or normal WT pancreas. c. Co-expression of MG53 with myc-KIF11 HEK293T cells significantly decreased KIF11 protein levels without 10 µM MG132 (a proteasome inhibitor) treatment, but had no effect in the presence of MG132, suggesting MG53-mediated down-regulation of KIF11 is not through proteasome degradation. d. qPCR was conducted to measure KIF11 mRNA levels in PANC-1 cells infected with Ad-Ctrl or Ad-MG53 and treated with 1 µg/ml Dox, showing considerable increase in MG53 gene expression and decrease in KIF11 gene expression in PANC-1 cells 48 h after Ad-MG53 infection. Data are mean ± SEM; p values were calculated using unpaired Student's t-test and presented in the individual panels.

Fig. 3.

Fig. 3

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Overexpressing MG53 inhibited colony formation and proliferation of PANC-1 cells. a. Live cell imaging and quantitative analysis revealed that induced MG53 expression with Dox inhibited the growth of PANC-1 cells compared to Ad-Ctrl following infection at 48 and 72 h. b. The colony formation assay, conducted as described in the Methods section, revealed significantly fewer colonies in the group with inducible MG53 expression compared to the control group. Representative enlarged images showing the size of the colonies are provided in Supplemental Fig. S5. Data are mean ± SEM; p value was calculated by unpaired Student's t-test and presented in the individual panels.

Decreased MG53 and increased KIF11 expression in pancreatic cancer

Both qPCR and Western blots were conducted on human pancreatic cancer cells (MPanc-96 and PANC-1) [13,16] and normal pancreatic stellate cells (PaSCs) [16,24]. Results revealed elevated KIF11 gene and protein level in both MPanc-96 and PANC-1 cells compared to PaSCs, consistent with the recognized oncogenic role of KIF11 in pancreatic cancer [13,14], while MG53 levels was notably decreased in cancer cells, suggesting a potential negative correlation between MG53 and KIF11 expression in pancreatic cancer cells (Fig. 4a-b & Supplemental Fig. S2).

Fig. 4.

Fig. 4

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Decreased MG53 and increased KIF11 were found in human pancreatic cancer cell lines and PDAC biopsy samples. a-b. Western blots and quantitative analysis were performed on human pancreatic cancer cells (MPanc-96 and PANC-1) and normal pancreatic stellate cells (PaSCs), showing significantly higher KIF11 levels in cancer cells compared to PaSCs, while MG53 protein levels were significantly lower in cancer cells compared to PaSCs. c-d. Protein levels of MG53 and KIF11 were compared in pancreatic tumors and their adjacent normal tissue from human patients, revealing higher KIF11 expression and lower MG53 expression in tumors than in their adjacent normal tissues, with the ratio of KIF11 expression being significantly higher than that of MG53. Data are mean ± SEM; p values were calculated using one-way ANOVA with Tukey's multiple-comparison test (a), or unpaired Student's t-test (b), and presented in the individual panels.

The gene and protein levels of MG53 and KIF11 were also examined for pancreatic tumors versus their adjacent normal tissue from human patients (Fig. 4c & Supplemental Fig. S3). All pancreatic tumors showed higher expression of KIF11 compared with the adjacent normal tissues (Fig. 4d, left). Meanwhile, all pancreatic tumors exhibited lower expression of MG53 compared with adjacent normal tissues (Fig. 4d, middle). The ratio of KIFF11 to MG53 expression in tumors significantly exceeded that of adjacent normal tissues (Fig. 4d, right), further indicating the inverse relationship between MG53 and KIF11 in PDAC.

MG53 modulates KIF11 expression via transcriptional regulation, not as an E3 ligase

To gain insight into MG53’s modulation of KIF11, PANC-1 cells were infected with either Ad-MG53 or Ad-Ctrl and treated with doxycycline for induction of MG53 expression. Western blot showed that MG53 overexpression led to reduced expression of KIF11 (Fig. 5a), suggesting MG53’s role as a negative regulator of KIF11. Moreover, KIF11 expression was higher in pancreatic tumors derived from MG53-KO mice than from WT mice (Fig. 5b & Supplemental Fig. S4). Notably, while MG53 expression was undetectable in normal pancreas, its presence was observed in tumors from WT mice (but not from MG53-KO mice), indicating potential MG53 accumulation from circulation during tumor progression (Fig. 5b & Supplemental Fig. S4).

Our previous investigations have demonstrated MG53′s ability to regulate protein expression by functioning as an E3 ligase [25]. To determine if MG53 downregulates KIF11 through proteasome-mediated protein degradation, HEK293T cells were co-transfected with myc-tagged KIF11with or without MG53 for 24 h. Subsequently, cells were treated with or without 10 μM MG132 (a proteasome inhibitor) for 8 h before collecting cell lysates for Western blot analysis. As depicted in Fig. 5c, while HEK293T cells express endogenous KIF11 and undetectable levels of MG53, co-expression of MG53 with myc-KIF11 led to a significant reduction in KIF11 protein levels even without MG132. Moreover, the presence of MG132 did not affect the decrease in KIF11 induced by MG53 (lane 5 vs. lane 3), suggesting that the reduction of KIF11 by MG53 does not occur through proteasome-dependent protein degradation.

Following this, quantitative polymerase chain reaction (qPCR) analysis was conducted to quantify KIF11 mRNA levels in PANC-1 cells infected with either Ad-Ctrl or Ad-MG53 in the presence of 1 µg/ml Dox (Fig. 5d). We observed a remarkable over 50-fold increase in MG53 gene expression in PANC-1 cells following 48 h of Ad-MG53 infection, accompanied by a significant reduction in KIF11 gene expression (Fig. 5d). Similar results were observed when overexpressing MG53 in other human pancreatic cell lines (MPanc-96 and PAN2, data not shown). These results are consistent with the changes observed in MG53 or KIF11 protein levels (Fig. 5a), indicating that MG53 likely downregulated KIF11 expression through transcriptional regulation.

The nuclear translocation of MG53 in human pancreatic cancer

Immunostaining of MG53 was performed on PANC-1 cells infected with either Ad-Ctrl or Ad-MG53. While PANC-1 cells typically exhibit low endogenous MG53 protein levels (Fig. 4a), immunofluorescence imaging revealed a predominant nuclear localization of MG53 in Ad-Ctrl infected cells (Fig. 6a, left, indicated by yellow arrow). Following infection with Ad-MG53, there was a noticeable increase in MG53 intensity within the nuclei of PANC-1 cells (Fig. 6b, right, indicated by yellow arrow).

Fig. 6.

Fig. 6

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Nuclear translocation of MG53 in pancreatic cancer cells. a. Staining PANC-1 cells with MG53 revealed low levels of endogenous MG53 protein, with the majority of MG53 signal observed within the cell nuclei. b. After infecting PANC-1 cells with Ad-MG53, overexpressed MG53 leads to significantly intensified signals observed within the nuclei of PANC-1 cells. c. Cell fractionation of PaSC and PANC-1 cells infected with Ad-Ctrl or Ad-MG53, using GAPDH to define cytosolic fractions and DOT1L to define nuclear fractions, revealed significant protein level of MG53 in nuclear fractions and decreased KIF11 levels in both cytosolic and nuclear fractions upon MG53 overexpression. d. Confocal imaging of human pancreatic tumor sections, stained with antibodies against MG53 and pan-cytokeratin (a marker of cancer cells), revealed MG53 signals primarily in non-cancerous cells and stromal cells; however, upon enlargement, some MG53 signals were detected in the nuclei of pan-cytokeratin positive PDAC cells.

Cell fractionation was performed on PaSC and PANC-1 cells infected with Ad-Ctrl or Ad-MG53 in the presence of doxycycline, followed by Western blot analysis for MG53 and KIF11 (Fig. 6c). GAPDH and DOTIL were utilized as a marker for cytosolic and nuclear fractions, respectively. Consistent with the immunofluorescence findings (Fig. 6b), Western blot analysis showed an abundance of MG53 protein within the nuclear fraction of Ad-MG53 infected PANC-1 cells. Conversely, the expression of KIF11 was markedly reduced in both cytosolic and nuclear fractions upon MG53 overexpression in both PaSC and PANC-1 cells.

Confocal imaging was then performed on sections of human pancreatic tumors after immuno-fluorescent staining with antibodies targeting MG53 (green) and pan-cytokeratin (red, serving as a marker for pancreatic cancer cells). While most of the MG53 signal was localized in non-cancerous cells such as stromal cells, some MG53 signals were detected in the nuclear regions of pan-cytokeratin-positive PDAC cells upon magnification of the images (Fig. 6d, indicated by yellow arrows). Confocal images were also collected from human tumor sections with immunofluorescent staining for KIF11 and pan-cytokeratin (Supplemental Fig. S5). These images revealed that KIF11 signals were primarily localized in cancer cells that showed positive staining for pan-cytokeratin. These findings support the presence of MG53 within the nuclei of PDAC cells, suggesting its involvement in modulating KIF11 expression and exerting a suppressive effect on cell proliferation.

Overexpressing MG53 decreased the number of mitotic cells in PANC-1 cells

PANC-1 cells infected with Ad-Ctrl exhibited positive staining of KIF11, with a substantial proportion of mitotic cells displaying distinct KIF11 nuclear staining of spindle structures (Fig. 7a, left, indicated by white arrow). However, KIF11 fluorescence intensity was notably reduced in PANC-1 cells infected with Ad-MG53 (Fig. 7a, middle), and the number of mitotic cells significantly decreased upon incubation with Dox for 48 h to induce MG53 overexpression (Fig. 7a, right), indicating suppressed mitosis resulting from MG53 overexpression.

Fig. 7.

Fig. 7

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MG53 suppresses pancreatic cancer cell mitosis. a. Immunostaining of KIF11 in PANC-1 cells infected with Ad-Ctrl or Ad-MG53 revealed that while Ad-Ctrl-infected cells showed positive staining and numerous mitotic cells with clear KIF11 nuclear staining, KIF11 fluorescence decreased significantly and mitotic cell numbers reduced upon Dox-induced MG53 overexpression, indicating suppressed mitosis. b. Immunostaining showed lower expression of KIF11 in normal pancreas and tumors from WT mice compared to tumors from MG53-KO mice, with a significantly higher number of KIF11-positive mitotic cells observed in tumors from MG53-KO mice compared to that from WT mice. Data are mean ± SEM; p values were calculated using unpaired Student's t-test (a) or one-way ANOVA with Tukey's multiple-comparison test (b) and presented in the individual panels.

Immunostaining of KIF11 conducted on pancreatic tissue demonstrated low expression of KIF11 in WT pancreas, contrasting with high expression observed in tumors collected from MG53-KO mice (Fig. 7b). Of significant note, the number of KIF11-positive mitotic cells detected in tumors from MG53-KO mice was markedly higher than that observed in WT mice (Fig. 7b, indicated by red arrow). These findings collectively reinforce our hypothesis that circulating MG53 exerts a crucial role in suppressing pancreatic tumorigenesis.

MG53 interacts with the promoter of KIF11 to suppress its transcription activity and the cell cycle

To elucidate the mechanism underlying MG53’s transcriptional regulation of KIF11 expression, ChIP assays were conducted following the manufacturer's protocol. PANC-1 cells were infected with Ad-Ctrl or Ad-MG53 in the presence of 1 µg/ml Dox for 48 h, followed by cross-linking and sonication. Immunoprecipitation using an MG53 antibody or control IgG was performed to isolate chromatin/DNA fragments, which were then quantified using qPCR. The results revealed that MG53 binds to the promoter regions of KIF11 in PANC-1 cells (Fig. 8a). Furthermore, the promoter region of KIF11 was cloned into a luciferase reporter construct (KIF11-p-Luc) and co-transfected with MG53 or a vector control in HEK293T cells. Luciferase gene transcription under the control of the KIF11 promoter was significantly suppressed by MG53, indicating that MG53 effectively interacts with the promoter of KIF11 to inhibit its transcriptional activity (Fig. 8b).

Fig. 8.

Fig. 8

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MG53 binds to the KIF11 promoter, suppressing its gene expression and inhibiting pancreatic cancer cell proliferation by inducing G2/M phase cell cycle arrest. a. ChIP analysis showed that MG53 binds to the promoter regions of KIF11 in PANC-1 cells infected with Ad-Ctrl or Ad-MG53. b. Overexpressing MG53 significantly inhibited luciferase gene transcription controlled by the KIF11 promoter in HEK293T cells following co-transfection with KIF11-p-Luc and MG53, confirming its ability to effectively bind to and suppress the transcription activity of KIF11 promoter. c. Flow cytometry analysis following infection with Ad-MG53 and treatment with Dox revealed a significant increase in the number of PANC-1 cells in G2 phase and a decrease in cells in G0/G1 phase. d. Quantitative analysis for the results shown in panel c. e. Western blot analysis was conducted to examine the protein expression levels of MG53, KIF11, and cell cycle checkpoint proteins (CCNB1, phosphorylated or total CDC2) in human pancreatic PANC-1 cancer cells following infection with adenovirus expressing Ad-Ctrl or Ad-MG53. GAPDH was used as a loading control. Quantitative analysis was performed on three independent sets of experiments. Asterisk (*) indicates a p-value of <0.05 compared to Ad-Ctrl for each set of experiments. f. A schematic illustrates MG53′s anti-tumor function by suppressing KIF11 gene expression, thereby inhibiting cell mitosis and proliferation. Data are mean ± SEM; p values were calculated using unpaired Student's t-test and presented in the individual panels.

Additionally, cell cycle analysis was performed by staining PANC-1 cells with propidium iodide (PI) after 48 h of infection with Ad-Ctrl or Ad-MG53 and treatment with 1 µg/ml Dox. Flow cytometry analysis revealed that overexpression of MG53 significantly increased the population of PANC-1 cells in the G2 phase while reducing the number of cells in the G0/G1 phase (Fig. 8c and d). Western blot analysis of PANC-1 cells infected with adenovirus expressing Ad-Ctrl and Ad-MG53 indicated a reduced expression of Cyclin B1 and phosphorylated Cdc2 upon MG53-mediated down-regulation of KIF11. This further confirms the inhibition of cell cycle checkpoints through the overexpression of MG53 in PANC-1 cells (Fig. 8e).

Collectively, these findings support the concept that MG53 suppresses the growth and proliferation of pancreatic cancer cells by interacting with the promoter of KIF11 and transcriptionally suppressing the expression of KIF11, thereby inducing cell cycle arrest and inhibiting cell proliferation (Fig. 8f).

Discussion

MG53/TRIM72, initially identified as a key molecule involved in tissue repair, has been extensively studied for its functions within the cytosol or vesicles, particularly in tissue repair and anti-inflammatory processes. However, its potential roles within the cell nucleus have remained unclear. In this study, we present novel findings demonstrating that MG53 exerts a suppressive effect on pancreatic cancer cell proliferation through its nuclear activity, specifically by transcriptionally inhibiting the expression of the KIF11 gene.

The rationale for examining the association between MG53 and KIF11 in pancreatic cancer in this study is based on the following facts: First, KIF11 has been suggested to play an important role in promoting the proliferation of pancreatic cancer cells [13,14]. Therefore, targeting the hyperactivity of KIF11 through molecular approaches presents a promising avenue for PDAC therapy. Second, results from our group and other researchers have demonstrated that MG53 deficiency promotes tumor growth and metastasis in lung cancer [9], suggesting a potential tumor suppressor role for MG53. Additionally, decreased MG53 levels in the bloodstream have been associated with an elevated risk of cancer development in human patients [10]. Our recent findings also indicated that MG53 acts as a tumor suppressor by targeting G3BP2 or stress granule activity in non-small cell lung cancer [11]. Therefore, we hypothesized that MG53 might suppress cancer cell proliferation by down-regulating KIF11 expression. Third, our current results indicate an inverse association between MG53 and KIF11 in both cancer cell lines and pancreatic tumors collected from PDAC patients. Furthermore, we demonstrated that MG53 can bind to the promoter of KIF11 and transcriptionally inhibit its gene expression, leading to cell cycle arrest and the inhibition of cancer cell proliferation.

Our biochemical assay revealed that the expression of MG53 is undetectable in both KPC-Luc cells and normal pancreas from WT mice. However, MG53 presence was observed in pancreatic tumors collected from WT mice, whereas no MG53 was detectable in tumors from MG53-KO mice. One plausible explanation is that MG53 translocated to the tumor site from the circulatory system. Previous studies have demonstrated the presence of fluorescence-labeled recombinant human MG53 protein (rhMG53) at injury sites in the kidney and liver following stress-induced organ injury and intravenous injection of rhMG53, despite the absence of endogenous MG53 expression in healthy kidney and liver tissues under normal conditions [4,26]. Another potential source of MG53 in pancreatic tumors could be immune cell infiltration, as previous observations indicate low levels of MG53 in macrophages [27]. Future investigations are necessary to elucidate the origin of MG53 protein in pancreatic tumors developing in WT pancreases.

Prior research conducted by our team has elucidated the significant involvement of MG53 in tissue injury and subsequent repair processes in different normal organs [3,4,6,26,28]. Furthermore, investigations have revealed a systematic decline in MG53 levels within vital organs observed in both aging mice and humans [8]. Consequently, the safety profile and therapeutic efficacy of administering rhMG53 have undergone extensive evaluation across diverse disease models [8,26,[29], [30], [31]]. This study presents compelling in vivo evidence demonstrating that the systemic presence of MG53 effectively inhibits pancreatic tumor growth in both models of orthotopic transplantation and subcutaneous xenograft. Additionally, our in vitro findings reveal that MG53 overexpression suppresses the proliferation of human pancreatic cancer cells. Our mechanistic investigations suggest that MG53′s anti-tumor activity may involve the transcriptional down-regulation of KIF11 gene expression, a pivotal molecule in cell mitosis regulation. Notably, increased KIF11 expression has been correlated with the advancement and metastasis of various cancers, including pancreatic cells [13,14]. In fact, pharmaceutical inhibition of KIF11 has been proposed [14,32], with several KIF11 inhibitors undergoing clinical testing for PDAC treatment. However, it is important to acknowledge that like other chemotherapy drugs [18,33], the use of KIF11 inhibitors may induce toxicity to heart or other vital organs due to their nature as small molecule chemicals. Hence, future studies focusing on systematically comparing the advantages and disadvantages of rhMG53 versus KIF11 inhibitors would yield valuable insights for optimizing therapeutic strategies for PDAC management.

In our prior investigation regarding the role of MG53 in lung cancer, we observed that targeted upregulation of MG53 expression in non-small cell lung cancer cells effectively inhibits lung cancer growth [11]. This inhibition occurs through the physical interaction and subsequent degradation of G3BP2, resulting in reduced formation of stress granules in a xenograft model. Additionally, recent findings by Fang et al. have underscored MG53′s tumor suppressive function in colorectal and gastric cancer, demonstrating its ability to facilitate cyclin D1 degradation [34]. Our mechanistic analysis revealed that MG53 can translocate into cancer cell nuclei, where it binds to the promoter region of KIF11, thereby downregulating its gene expression and halting cell mitosis, ultimately impeding cancer cell proliferation. Interestingly, our data indicates that MG53-mediated reduction in KIF11 expression is not reliant on MG53′s E3 ligase activity, as inhibiting proteasome activity did not attenuate the decrease in KIF11 levels observed in vitro.

Conclusions

In summary, our study utilizing syngeneic orthotopic transplantation model elucidated the pivotal role of MG53 as a tumor suppressor in pancreatic cancer. The heightened tumor growth observed in MG53-KO mice, along with the aggressive cancer cell proliferation in subcutaneous transplant, underscores the significance of MG53 in inhibiting pancreatic tumor progression. Our findings reveal MG53-mediated transcriptional downregulation of KIF11, leading to cell cycle arrest and inhibition of cancer cell proliferation. These insights offer a promising avenue for developing novel therapeutic strategies targeting MG53-KIF11 axis in PDAC, potentially improving clinical outcomes for PDAC patients.

List of abbreviations

ATCC: American Type Culture Collection

CRISPR: Clusters of Regularly Interspaced Short Palindromic Repeats cDNA: Complementary DNA

DMEM: Dulbecco's Modified Eagle's Medium

Dox: Doxycycline

FBS: Fetal Bovine Serum

IACUC: Institutional Animal Care and Use Committee

IHC: Immunohistochemistry

IRB: Institutional Review Board

KIF11: kinesin family member 11

KO: Knock-out

KPC: LSL-KRasG12D/LSL-Trp53−/−/Pdx1-Cre;

KPC-Luc: murine pancreatic cancer cells derived from KPC mice and expressing enhanced firefly luciferase.

MG53: Mitsugumin 53, also known as TRIM72

PBS: Dulbecco's Phosphate Buffered Saline

PDAC: Pancreatic ductal adenocarcinoma

PCR: Polymerase Chain Reaction

PFA: Paraformaldehyde rhMG53: Recombinant human MG53 (protein)

TRIM: Tripartite Motif

WT: Wild Type

Declarations

Ethics approval and consent to participate

All animal care and usage were done in accordance with federal policies and guidelines and approved by IACUC at The Ohio State University and University of Virginia.

Consent for publication

Not applicable.

Funding

This work was supported by the National Institute of Health (R01HL157215 to J.M. and C.C., R01AG071676 to J.M., R01CA214865 to A.T.).

CRediT authorship contribution statement

Xiao-Liang Wang: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Xiangfei He: Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis, Data curation. Tong Gao: Data curation, Methodology. Xinyu Zhou: Data curation, Formal analysis, Methodology. Zobeida Cruz-Monserrate: Writing – review & editing, Methodology, Conceptualization. Allan Tsung: Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization. Jianjie Ma: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. Chuanxi Cai: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

Dr. Jianjie Ma (J.M.) is founder of TRIM-edicine, which develops MG53 for the treatment of human disease. Patents on the use of MG53 are held by Rutgers University-Robert Wood Johnson Medical School and The Ohio State University.

Acknowledgements

We thank Drs. Dan Stover and Nancy Single for their help on the collection of human pancreatic biopsy, which research was supported by the Department of Pathology's Tissue Procurement Service, The Ohio State University Wexner Medical Center, Columbus, OH.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102118.

Contributor Information

Jianjie Ma, Email: Jianjie.Ma@virginia.edu.

Chuanxi Cai, Email: Chuanxi.Cai@virginia.edu.

Appendix. Supplementary materials

mmc1.pdf (1.7MB, pdf)

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Supplementary Materials

mmc1.pdf (1.7MB, pdf)

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