KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway

doi:10.21203/rs.3.rs-4838885/v1

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Research Article

KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway

https://doi.org/10.21203/rs.3.rs-4838885/v1

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Background: Pancreatic cancer (PC) is a highly lethal tumor of the gastrointestinal tract. New molecular targets are urgently needed for its treatment. Kinesin family member C1 (KIFC1) is implicated in the development and progression of several types of cancer. Previously, our studies indicated that KIFC1 is overexpressed in hepatocellular carcinoma and activates the malignant behavior of hepatocellular carcinoma through the PI3K/AKT pathway. However, the molecular and functional mechanisms of KIFC1
in PC have not been investigated.

Methods: In this study, high-throughput sequencing technology was utilized to characterize differential gene expression profiles in patients with PC. KIFC1 was revealed by screening up-regulated genes from our sequenced data and the Gene Expression Omnibus (GEO) database. Sixty-two PC tissues were analyzed to determine
the correlation of KIFC1 expression with the clinicopathological features and prognosis
of patients. The role of KIFC1 in proliferation, migration and invasion in PC was verified both in vitro and in vivo. Bioinformatics analysis, coimmunoprecipitation (CoIP), and western blotting were performed to identify proteins that interact with KIFC1and further affect the downstream pathway.

Results: According to high-throughput sequencing and the GEO database, KIFC1 is highly expressed in PC. KIFC1 is highly expressed in PC tissues and cells and is positively correlated with poor patient prognosis and malignant cellular behavior. Silencing KIFC1 inhibited the proliferation, migration, and invasion of PC cells, and
overexpression of KIFC1 had the opposite effect. Protein‒protein interaction (PPI) and
Co-IP analyses indicated that KIFC1 interacts with and regulates BUB1. Overexpression of BUB1 can also promote the proliferation, migration, and invasion of PC cells. BUB1 acts as an intermediary in the activation of the Wnt/β-catenin pathway by KIFC1, leading to an increase in the malignant behaviors of PC cells. The reversal of Wnt/β-catenin activation and increase in cellular malignant behavior induced by KIFC1 overexpression are achieved by silencing BUB1. These biological functions of KIFC1 in PC were also confirmed in a nude mouse xenograft model.

Conclusions: Our experiments demonstrated for the first time that KIFC1 can influence PC progression by regulating BUB1 to activate the Wnt/β-catenin pathway. Therefore, KIFC1 shows promise as an attractive therapeutic target for PC in the future.

KIFC1

BUB1

Pancreatic cancer

Protein interactions

Tumorigenesis

Pancreatic cancer (PC) causes approximately 50,000 deaths each year in America. Unfortunately, most PC patients have a poor prognosis. Investigations have shown that the 5-year survival rate for patients with PC is only 13%[1]. Surgical removal of the lesion is currently the only viable option for eradicating PC, but early metastasis or extensive local invasion renders resection for PC patients less than 20%[2, 3]. Although adjuvant and systemic chemotherapy are currently indispensable therapeutic methods for improving long-term outcomes, their effects are still unclear[4].

In 1985, Ronald D. Vale discovered kinesin in the squid giant axon, which induces microtubule-based movements[5]. To date, 14 distinct families of kinesin superfamily proteins (KIFs) have been identified, ranging from KIF1 to KIF14[6]. KIFs participate in a range of cellular processes, including synaptic vesicle transport, centrosome clustering, and chromosomal transport during mitosis or meiosis, due to their function as molecular motors[7, 8]. Kinesin family member C1 (KIFC1) is a member of the kinesin-14 family of C-type kinesins. Previous research has shown that KIFC1 is linked to the development and progression of several types of tumors, including prostate carcinoma[9], breast carcinoma[10], gastric carcinoma[11], endometrial carcinoma[12], and non-small cell lung carcinoma[13]. Our previous experiments showed that KIFC1 is significantly overexpressed in hepatocellular carcinoma and contributes to the progression of the disease, resulting in a poor prognosis[14]. However, the molecular and functional mechanisms of KIFC1 in PC have not been reported.

The BUB1 protein plays a crucial regulatory role in mitosis, as demonstrated by recent studies[15]. It encodes a protein kinase that activates the spindle checkpoint by phosphorylating members of the mitotic checkpoint complex[16–18]. Various types of cancer, including bladder[19], pancreatic[20], and breast cancers[21], have been linked to mutations in BUB1. Kenneth B. Kaplan et al. reported that BUB1 phosphorylates the adenomatosis polyposis coli (APC) protein in the WNT pathway[22]. Phosphorylated APC proteins may lose their ability to inhibit the WNT pathway. Similarly, no studies have reported on the specific molecular mechanisms and functions of BUB1 in PC.

The Wnt signaling pathway was initially identified in a mouse mammary tumor virus model and was designated Int-1 by Roel Nusse[23]. The Wnt/β-catenin signaling pathway is a canonical signaling pathway in which the β-catenin protein plays a key role. In the nucleus, β-catenin binds to DNA-binding proteins of the T-cell factor/lymphoid enhancing factor (TCF/LEF) family, leading to the activation of downstream target genes. Wnt pathway activation is inhibited by the degradation of β-catenin, which is phosphorylated by a disruption complex composed of APC, Axin, and GSK3β. This phosphorylation recruits the E3 ubiquitin ligase containing β-TrCP to degrade β-catenin. [24].

However, the molecular interactions between the KIFC1, BUB1, and Wnt signaling pathways in PC have not been fully elucidated. Our study revealed that KIFC1 interacts with downstream BUB1, ultimately activating the Wnt pathway and promoting PC development. This study provides a new molecular mechanism of KIFC1 in PC development and identifies new targets for the future treatment of PC.

Patients and tissue samples

This study included sixty-two patients with PC who were pathologically diagnosed between 2016 and 2023 and who underwent pancreatectomy at the Department of Hepatobiliary Surgery of the Second Affiliated Hospital of Nanchang University. The specimens were subjected to immunohistochemical analysis after being embedded in formalin and paraffin. The patients' clinicopathological data were also collected. All patients provided informed consent. Tumor staging was determined according to the Union for International Cancer Control TNM classification guide (8th edition, 2019). The Ethics Committee of the Second Affiliated Hospital approved the study.

Cell lines

PANC-1, SW-1990, BXPC-3, ASPC-1, and H6C7 (human normal pancreatic ductal epithelial cells) cells were purchased from the Institutes for Life Sciences, Chinese Academy of Sciences (Beijing, China). All cells were cultured in DMEM (Thermo Fisher, 12430054, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, SH30396.02, USA), except for the ASPC-1 cells, which were cultured in RPMI-1640 medium (Thermo Fisher, 11875119, USA) supplemented with 10% FBS in a humidified incubator supplemented with 5% carbon dioxide at 37°C, and the medium was changed every 3 days.

Immunohistochemistry

Tissue specimens were dewaxed in xylene and rehydrated in a graded series of ethanol. Afterwards, the tissue sections were placed in a pressure cooker at 100°C for 15 minutes to repair the antigens. Subsequently, the sections were incubated with H₂O₂ for 15 minutes at room temperature to block endogenous peroxidase activity. Next, the sections were blocked with goat serum (Thermo Fisher, 16210064, USA) for 30 minutes. Next, the sections were incubated with an anti-KIFC1 antibody (1:200, ORIGENE, TA38608) at 4°C overnight. Afterwards, the sections were incubated with the corresponding secondary antibodies. Both the staining intensity and area of positive staining for KIFC1 were evaluated by two pathologists in a mutually blinded manner. Staining was graded as 0 (negative), 1 (weakly positive), 2 (moderate) or 3 (strongly positive) based on the intensity of staining. The extent of staining was scored as 1 (< 10%), 2 (10–40%), 3 (40%-75%) or 4 (> 75%). The intensity and extent of the staining were multiplied to obtain a total staining score. The KIFC1 score ranged from 0 to 12, which allowed the specimens to be categorized into a low-expression group (0–3) and a high-expression group (3–12).

Data mining and bioinformatics analysis

The expression profiles of KIFC1 and BUB1 in PC and their respective survival curves were obtained from the Gene Expression Profiling Interactive Analysis (GEPIA) online database (http://gepia.cancer-pku.cn/). KIFC1 and BUB1 gene correlation analyses were performed with the Gene_Corr module of the TIMER2.0 database (http://timer.cistrome.org/). The PPIs were first identified from the STRING database and then mapped using Cytoscape v3.9.0 software. The GSE107160, GSE16515 and GSE15471 datasets were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/).

Cell transfection

Small interfering RNA (siRNA) targeting KIFC1 and lentiviruses for the knockdown or overexpression of KIFC1 and BUB1 were purchased from General Biol (Anhui, China). Small interfering RNA (siRNA) was used to grow the cells in 6-well plates to 70–80% confluence, and the cells were transfected with Lipofectamine 2000 (Invitrogen, 11668-019, USA) according to the manufacturer's protocol. After the cells reached 90% confluence, they were infected with lentivirus, followed by screening with puromycin to obtain stable overexpression and knockdown cell lines. The sequences of the siRNAs used were as follows: siRNA1: 5'-GGACUUAAAGGGUCAGUUATT-3' and siRNA2: 5'-CGGGAACGCCUUCGGGAAATT-3'.

Quantitative real-time PCR (qRT‒PCR)

The expression of KIFC1 was assessed by qRT‒PCR. Total RNA was isolated from PC cells using TRIzol reagent, and cDNA was obtained by reverse transcription according to the instructions of TRAN One-Step gDNA Removal and cDNA Synthesis SuperMix Kits (TransGen, AE311, China). qRT‒PCR was subsequently performed using SYBR® Premix Ex Taq II Kits (Takara, RR420B, USA). The 2^−ΔΔCt method was used to calculate KIFC1 using GAPDH as an internal reference[25] for relative expression. The sequences of the primers used were as follows: KIFC1-F: 5'-GCAGGAACTCAAGGGCAA-3', KIFC1-R 5'-GCTAAGGCGGGGTTGGAG-3'; and GAPDH-F: 5'-GGACCTGACCTGCCGTCTAG-3', GAPDH-R 5'-GTAGCCCAGGATGCCCTTGA-3'.

Western blotting

Cell or tissue samples were lysed using RIPA lysis buffer supplemented with protease inhibitors. The proteins were separated by SDS‒PAGE using either an 8% or 10% gel and then transferred to 0.22 \(\:\mu\:m\) PVDF membranes (Millipore, ISEQ00010, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4°C. The strips were then washed with TBST and incubated with secondary antibodies for one hour at room temperature. Finally, the strips were visualized using a bioimaging system and analyzed with ImageJ software.

Primary antibodies against KIFC1 (cat. no. TA384608, ORIGENE), BUB1 (cat. no. HA60053, HUABIO), β-catenin (cat. no. #8480, Celling Signaling), TCF-4 (cat. no. #2569, Celling Signaling), c-Myc (cat. no. #9402, Celling Signaling), cyclin D1 (cat. no. #55506, Celling Signaling), N-cadherin (cat. no. #13116, Celling Signaling), E-cadherin (cat. no. #3195, Celling Signaling), vimentin (cat. no. #5741, Celling Signaling), and GAPDH (cat. no. #5174, Celling Signaling) were used.

Cell Counting Kit-8

The transfected cells were inoculated into 96-well plates at a density of 4000 cells per well and cultured for 24, 48, or 72 hours. Then, 10 \(\:\mu\:L\:\)of CCK-8 (Abcam, ab228554, USA) reagent was added to each well at the end of each incubation cycle. After a 2-hour incubation, the absorbance of each well at 450\(\:\:nm\:\)was measured using a microplate reader.

Colony Formation Assay

Cells that had been transfected and resuspended were inoculated into six-well plates at a density of 800 cells per well. The cells were cultured for 14 days, and the cells in each well were fixed with paraformaldehyde for 30 minutes at room temperature and then stained with crystal violet for 10 minutes. A microscope was used to count the number of cell clusters.

Cell proliferation assay

Cell proliferation was assessed by a 5-ethynyl-2ʹdeoxyuridine (EdU) proliferation assay, and an EdU kit (UElandy, C6044S, China) was purchased from UElandy Biotechnology Co. in Suzhou, China. Transfected and resuspended cells were inoculated into 96-well plates at 1 × 10⁵ cells per well. Once the cells had attached to the wall, the EdU reagent was added to the medium, and the cells were incubated for two hours. After this, the cells were fixed with 4% paraformaldehyde and destained with glycine, and the cell membrane was permeabilized with 0.5% Triton X-100. YF594 or YF488 was added for 30 minutes at 25°C in the dark. After washing, Hoechst 33342 was added for another 30 minutes under the same conditions. Images were acquired using a fluorescence microscope.

Wound healing

The fused cells were treated with mitomycin (1\(\:\:\mu\:g/mL\)) for one hour after being switched to serum-free medium. Vertical scratches were made at the bottom using a 200 \(\:\mu\:l\:\)pipette gun tip. The shed cells were washed away with PBS, and the scratches were imaged at 0 h. Images were captured again 24 h after incubation, and the rate of cell migration was calculated.

Transwell assay

For the invasion assay, Matrigel was spread into Transwell chambers. Transfected cells were then resuspended in serum-free DMEM and added to the upper chambers, while DMEM containing 10% FBS was added to the lower chambers. The chambers were incubated for 48 hours in a 24-well plate. After incubation, the chambers were fixed with 4% paraformaldehyde for 25 minutes and stained with crystal violet for 5 minutes. Images were captured using a microscope, and the cells were counted.

Cell Cycle Assay

A total of 1×10⁵ posttransfection cells were collected and fixed with precooled 70% ethanol in a refrigerator at 4°C overnight. The following day, the cells were washed twice with PBS, stained with 0.5 ml of PI/RNase (Abcam, ab112116, USA) and resuspended. The cells were incubated for 30 minutes at room temperature in the dark. Afterward, the cell cycle distribution was analyzed using flow cytometry.

Animal Experiments

Female BALB/c nude mice (6–8 weeks old) weighing 15 ± 1 g from Beijing SPF Biotechnology Co., Ltd., were used to construct a xenograft tumor model. All animal experiments were conducted at Nanchang Royo Biotech Co,. Ltd. The aim of this study was to investigate the effect of KIFC1 on tumor formation in vivo.

The mice were injected subcutaneously with 1×10⁶ PANC-1 cells (with stable KIFC1 knockdown) resuspended in 100\(\:\:\mu\:L\:\)of serum-free DMEM. Tumor volumes were measured at five-day intervals, and images of the mice were captured after 30 days using a small animal live imager. After the mice were euthanized, the tumor tissue was removed for detection. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co,. Ltd (Nanchang, China, IACUC No. RYE2024033001)

Statistical analysis

All analyses were performed using SPSS 26.0 software. Survival curves were plotted using the Kaplan‒Meier method and compared using the log-rank test, and the association between KIFC1 and clinicopathology was tested using the \(\:\chi\:2\) test. Bivariate correlations were calculated using Pearson's correlation coefficient. Comparisons between two groups were made using two independent samples t tests, and differences were considered statistically significant at P < 0.05. The data are presented as the means ± SDs, and P < 0.05 was considered to indicate statistical significance.

KIFC1 is highly expressed in PC tissue and is associated with advanced clinical stage and poor prognosis.

Both the high-throughput sequencing and GSE107610 datasets indicated that KIFC1 is highly expressed in PC (Fig. 1A). To explore the expression and location of KIFC1 in PC patients, we performed immunohistochemistry (IHC) analysis on 62 PC patient samples. We observed almost no KIFC1 expression in normal tissues. On the other hand, KIFC1 expression was significantly high in neoplastic tissues, and it was detected mainly in the nuclei of tumor cells (Fig. 1B). This finding was supported by findings obtained from the online GEPIA database (Fig. 1C). Furthermore, by analyzing the clinicopathological features of 62 patients, we found that a high KIFC1 expression level was closely correlated with T stage (P = 0.016), TNM stage (P = 0.017) and histological grade (P = 0.001) in patients with PC (Table 1). These findings indicate that KIFC1 expression is clearly related to poor clinical and pathological stage. Therefore, we analyzed the association between KIFC1 expression and patient survival by Kaplan‒Meier analysis, and the results demonstrated that KIFC1-high PC patients had a significantly worse overall survival (OS) than did KIFC1-low patients (log rank P = 0.008; Fig. 1E). This result was also confirmed by the GEPIA database (log rank P = 0.0059; Fig. 1D).

Table 1

Relationships between KIFC1 expression and the clinicopathological features of 62 PC patients.
Variables	n	KIFC1		χ²	P value
Variables	n	High expression	Low expression	χ²	P value
Sex				0.466	0.495
Male	25	17(68.0%)	8(32.0%)
Female	37	22(59.5%)	15(40.5%)
Age (years)				0.726	0.394
≥ 60	34	23(67.6%)	11(32.4%)
< 60	28	16(57.1%)	12(42.9%)
T staging				8.290	0.016*
T1	20	8(40.0%)	12(60.0%)
T2	27	18(66.7%)	9(33.3%)
T3	15	13(86.7%)	2(13.3%)
Nerve infiltration				0.777	0.378
Yes	47	31(66.0%)	16(34.0%)
No	15	8(53.3%)	7(46.6%)
Venous invasion				2.503	0.114
Yes	35	25(71.4%)	10(28.6%)
No	27	14(51.9%)	13(48.1%)
Histological grade				11.519	0.001*
High	19	6(31.6%)	13(68.4%)
Low / Medium	43	33(76.7%)	10(23.3%)
Lymph node status				0.521	0.470
Negative	26	15(57.7%)	11(42.3%)
Positive	36	24(66.7%)	12(33.3%)
TNM staging				8.127	0.017*
I	19	7(36.8%)	12(63.2%)
II	29	21(72.4%)	8(27.6%)
III	14	11(78.6%)	3(21.4%)

KIFC1 overexpression promotes PC cell growth in vitro

The KIFC1 protein and mRNA were examined by western blotting and qRT‒PCR, and the results revealed that KIFC1 was more highly expressed in PC cell lines than in normal pancreatic ductal epithelial cells (Fig. 1F and G). To confirm that KIFC1 promotes PC cell proliferation, two independent siRNAs and one KIFC1-overexpressing lentiviral vector were used to silence or overexpress KIFC1, respectively. SiRNA transfection and lentivirus infection were detected in PANC-1 and SW-1990 cells via western blotting, and the KIFC1 protein levels decreased and increased, respectively (Fig. 2A). Compared with control cells, SW-1990 cells overexpressing KIFC1 exhibited greater cell viability, colony formation and proliferation (Fig. 2B, C and 3A). However, KIFC1 knockdown in PANC-1 cells had opposite effects on cell viability, colony formation and proliferation (Fig. 2B, C and 3A). Furthermore, a cell cycle assay demonstrated that KIFC1 promoted progression through the cell cycle from the G1 to the S and G2 phases. (Fig. 3B).

KIFC1 promoted PC cell migration and invasion.

Scratch and Transwell assays were performed in PANC-1 and SW-1990 cells to examine the impact of KIFC1 on PC cell migration and invasion. A scratch assay indicated that KIFC1 overexpression markedly enhanced migration, while the opposite effect was observed in the control group (Fig. 4A). In addition, Transwell assays revealed that PC invasion capacity was closely related to KIFC1 expression (Fig. 4B). It is widely known that epithelial–mesenchymal transition (EMT) is an indispensable factor that promotes cell migration and invasion[26, 27]. Therefore, western blotting was used to verify the key role of EMT in PC cell migration and invasion. The results demonstrated that the epithelial marker E-cadherin was repressed; in contrast, the mesenchymal markers N-cadherin and vimentin were increased in KIFC1-overexpressing PC cells. However, silencing KIFC1 had the opposite effect (Fig. 4C). Overall, the above results indicated that KIFC1 promoted PC cell migration and invasion.

KIFC1 overexpression activated the Wnt/β/catenin pathway in PC cells.

In a previous study, the Wnt/β/catenin pathway was verified to contribute to PC malignancy[28]. The dephosphorylation of β-catenin prevents it from being degraded by ubiquitination, after which it accumulates in the nucleus and increases the expression of TCF/LEF. TCF/LEF subsequently interacts with downstream targets, such as c-Myc and cyclin D1[29]. Western blotting revealed that KIFC1 overexpression upregulated β-catenin, TCF4, c-My and cyclin D1, while KIFC1 depletion downregulated the expression of these genes (Fig. 4C). Thus, the results indicated that KIFC1 might affect PC malignancy by activating the Wnt/β-catenin signaling pathway.

BUB1 interacts with KIFC1 at the protein level, is highly expressed in PC patients and is associated with poor prognosis.

Using high-throughput sequencing, we screened 2922 upregulated genes. After genes that were not interrelated were eliminated, PPI analysis revealed that KIFC1 may interact with BUB1. (Fig. 5A). To investigate the relationship between KIFC1 and BUB1 in PC, we analyzed transcriptomic data from The Cancer Genome Atlas (TCGA) Spearman's correlation analysis confirmed a strong positive correlation between the expression of KIFC1 and that of BUB1 in PC (r = 0.835; Fig. 5B). Moreover, interactions between KIFC1 and BUB1 were observed at the protein level via Co-IP assays (Fig. 5C). To confirm the relationship between BUB1 expression levels and clinical outcomes, we selected two datasets (GSE15471 and GSE16151) from the GEO database for paired-sample t tests. The results showed that BUB1 expression was significantly greater in tumor tissues than in adjacent normal tissues (Fig. 5D and E). Additionally, the GEPIA database revealed high expression of BUB1 in patients with tumors, which adversely affected patient prognosis (Fig. 5F and G).

BUB1 mediates the effect of KIFC1 on the Wnt/β-catenin pathway and the malignancy of PC cells.

Western blotting revealed that BUB1 was successfully knocked down or overexpressed (Fig. 6A). EdU experiments revealed that BUB1 knockdown decreased the proliferation of PC cells, whereas BUB1 overexpression had the opposite effect (Fig. 6B). Similarly, a positive correlation between BUB1 expression and cell migration and invasion ability was observed in the scratch and Transwell assays (Fig. 6C and D). The knockdown and overexpression of KIFC1 in PANC-1 cells resulted in a similar trend for BUB1 (Fig. 7A). A lentivirus was used to knock down BUB1, which reversed the positive effects of KIFC1 overexpression on the proliferation, migration, and invasion ability of PANC-1 cells (Fig. 7B-D). Similarly, decreased protein levels of β-catenin, TCF-4, c-Myc, and cyclin D1 were observed in KIFC1-overexpressing cells upon BUB1 silencing (Fig. 7E). These results demonstrated that reducing BUB1 expression inhibits the activation of the Wnt/β-catenin pathway induced by KIFC1 overexpression.

KIFC1 enhances PC growth and activation of the Wnt/β-catenin pathway in vivo

We established subcutaneous tumor models to explore the function of KIFC1 in regulating PC tumorigenesis. Tumors with silenced KIFC1 exhibited significantly lower growth rates, weights, and volumes than those in the control group. (Fig. 8A-C). Moreover, a small animal imaging system was used to evaluate tumor growth, and the results also verified that KIFC1 knockdown obviously suppressed cancer cell proliferation (Fig. 8D). Furthermore, Western blotting revealed that KIFC1 regulates BUB1 at the protein level and affects the activation of the Wnt/β-catenin pathway in vivo (Fig. 8E). Thus, in vivo experiments confirmed that KIFC1 regulates BUB1 and the downstream Wnt/β-catenin pathway, promoting tumor growth.

PC is a highly malignant tumor of the gastrointestinal tract, with an incidence rate almost equal to the mortality rate[30]. Due to its insidious early onset, most cases are already in advanced stages when it is detected. PC can only be eliminated through surgery, but the chances of recurrence after the procedure are high, leading to a poor prognosis[31]. Therefore, finding potential molecular targets for PC may lead to new directions for its treatment. Through a series of experiments, we confirmed that KIFC1 is highly expressed in PC cell lines and patients. KIFC1 overexpression activates the Wnt/β-catenin pathway through BUB1, promoting malignant behavior and functions in PC cells. These findings indicate that the KIFC1-BUB1-Wnt pathway signaling axis could be a more effective target for PC therapy.

With respect to KIFC1, growing evidence suggests that KIFC1 overexpression is strongly associated with cancer development, progression, and drug resistance[32, 33]. Cancer cells typically exhibit centrosome amplification, and these cells survive and proliferate by clustering supernumerary centrosomes for bipolar division[34, 35]. Coincidentally, KIFC1 can aggregate centrosomes to achieve bipolar division of cancer cells, promoting their survival and proliferation[36, 37]. These findings demonstrate that KIFC1 may promote PC progression by facilitating centrosome aggregation in PC cells.

Previous studies have demonstrated that the Wnt/β-catenin signaling pathway can promote PC tumorigenesis and drug resistance[38, 39]. Targeting this pathway has become a crucial initiative for treating PC and other gastrointestinal tumors[40]. In head and neck carcinoma, KIFC1 has been identified as an 'activator' of the Wnt/β-catenin signaling pathway, promoting the development of head and neck squamous cell carcinoma[41]. Our experimental results suggest that KIFC1 may promote PC development by activating the Wnt/β-catenin signaling pathway. Further mechanistic exploration revealed that KIFC1 binds to BUB1 at the protein level. Knocking down BUB1 reversed the increase in cell function and Wnt/β-catenin pathway activity caused by KIFC1 overexpression, suggesting that KIFC1 may activate the Wnt/β-catenin pathway via BUB1. BUB1 functions as a protein kinase that phosphorylates APC (a component of the disruption complex)[42]. We speculate that this phosphorylation may alter the activity of the disruption complex, preventing the degradation of most β-catenin. The retained β-catenin then enters the nucleus and binds to TCF/LEF proteins, transcribing downstream target genes. BUB1 is a crucial component in chromosome segregation, and BUB1 overexpression induces Aurora-B hyperactivation, resulting in chromosomal missegregation and aneuploidy. [43, 44]. In general, cancer cells with aneuploidy-containing and dispersed centromeres tend to undergo multipolarization, preventing them from dividing and leading to cell death[45]. However, KIFC1 promotes the aggregation of dispersed centrosomes in cancer cells, facilitating polar division and ensuring cell survival[36, 37]. The available evidence indicates that KIFC1 and BUB1 have molecular and cellular interactions that promote the proliferation, migration, and invasion of PC cells both in vitro and in vivo by activating the downstream Wnt/β-catenin pathway.

In summary, in the clinic, KIFC1 is expressed at high levels in PC tissues and cells, which significantly reduces the overall survival and results in a poor prognosis for patients. Mechanistically, KIFC1 overexpression contributed to PC cell proliferation, migration, and invasion via the BUB1/Wnt/β-catenin pathway. (Fig. 9).

KIFC1 Kinesin family member C1

PC Pancreatic cancer

TCGA The Cancer Genome Atlas Program

GEO Gene Expression Omnibus

APC Adenomatosis polyposis coli

GEPIA Gene Expression Profiling Interactive Analysis

PPI Protein‒Protein Interaction Networks

EMT Epithelial–mesenchymal transition

IHC Immunohistochemistry

TCF/LEF T-cell factor/Lymphoid enhancing factor

OS Overall Survival

Ethics Approval and Consent to Participate

The use of human tissue specimens for this study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Animal ethics were reviewed and approved by the Institutional Animal Care and Use Committee of Nanchang Royo Biotech Co., Ltd.

Competing interests

The authors declare no competing interests.

Consent for publication

All authors agreed with submission of the manuscript for publication and agree to be accountable for all aspect of the manuscript.

Funding

This research was funded by grants from the Natural Science Foundation of Jiangxi Province, China (No. 20232BAB216076, 20224BAB216050, 20232BAB206078 and 20224ACB206037); the Research Project of Education Department of Jiangxi Province, China (No. GJJ2200227 and GJJ2200237); the National Natural Science Foundation Incubation Project of The Second Affiliated Hospital of Nanchang University (2022YNF12020 and 2022YNFY12002); and the National Natural Science Foundation of China (No. 82360220 and 82360513).

Author Contribution

A.C: Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, and Writing—original draft. Y.X.Y: Resources, Validation, and Writing—original draft. J.Y.W and L.J.R: Resources, Data curation and Validation. Y.Q.Z: Resources, Funding acquisition and Writing—review & editing. Y.Z, R.X.L, M.Y.D and H.W: Resources, Validation, and Data curation. L.F: Resources, Supervision, Funding acquisition, Validation, and Writing—review & editing. X.W.F: Resources, Supervision, Funding acquisition and Writing—review & editing.

Acknowledgement

We would like to express our gratitude to the editors and reviewers for their hard work and the authors for their efforts.

Data availability

Data will be made available on request by contacting the corresponding author.

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KIFC1 overexpression promotes pancreatic carcinoma progression via the BUB1/WNT/β- catenin pathway

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Abstract

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Introduction

Materials and methods

Patients and tissue samples

Cell lines

Immunohistochemistry

Data mining and bioinformatics analysis

Cell transfection

Quantitative real-time PCR (qRT‒PCR)

Western blotting

Cell Counting Kit-8

Colony Formation Assay

Cell proliferation assay

Wound healing

Transwell assay

Cell Cycle Assay

Animal Experiments

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Results

KIFC1 overexpression promotes PC cell growth in vitro

KIFC1 enhances PC growth and activation of the Wnt/β-catenin pathway in vivo

Discussion

Conclusion

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