BCKDK Promotes Epithelial Ovarian Cancer Proliferation and Migration by Activating the MEK/ERK Signaling Pathway

Background: Ovarian cancer is the most fatal gynecologic cancer, and epithelial ovarian cancer (EOC) is the most common type. The branched-chain α-keto acid dehydrogenase kinase (BCKDK) plays an important role in many serious human diseases, including cancers. Its function in promoting cell proliferation and migration has been reported in various cancers. However, the biological role of BCKDK and its molecular mechanisms underlying EOC initiation and progression are unclear. Methods: First, the expression level of BCKDK in EOC cell lines or tissues was determined using tissue microarray (TMA)-based immunohistochemistry or western blotting. Then, growth curve analysis, anchorage-independent cell transformation assays, wound healing assays, cell migration assays, and tumor xenografts were used to test whether BCKDK could promote cell transformation or metastasis. Finally, the signaling pathways involved in this process were investigated by western blotting or immunoprecipitation. Results: We found that the expression of BCKDK was upregulated in EOC tissues and that high expression of BCKDK was correlated with an advanced pathological grade in patients. The ectopic overexpression of BCKDK promoted the proliferation and migration of EOC cells, and the knockdown of BCKDK with shRNAs inhibited the proliferation and migration of EOC ex vivo and in vivo. Moreover, BCKDK promoted EOC proliferation and migration by activating MEK. Conclusions: Our results demonstrate that BCKDK promotes EOC proliferation and migration by activating the MEK/ERK signaling pathway. Targeting the BCKDK-MEK axis may provide a new therapeutic strategy for treating patients with EOC.

Branched-chain α-keto acid dehydrogenase kinase (BCKDK) located in the mitochondrial matrix, belonging to pyruvate dehydrogenase kinases (PDKs) family 12 , which promoted the proliferation and metastasis of various tumors and was considered to be a strong therapeutic target for preventing tumors development [13][14][15][16][17][18] . Dysfunction of BCKDK was closely related to various human diseases, especially maple syrup urine disease. Bravo-Alonso and Oyarzabal found the excessive function of BCKDK resulted in maple syrup urine disease [19][20][21] . As diabetics had increased susceptibility to ovarian cancer, always divided into late stages when the rst diagnosis and had a poor prognosis 22 . Over-expression of BCKDK resulted in branched-chain amino acid (BCAA) increase, elevated plasma levels of BCAA were associated with a greater than 2-fold increased risk of future pancreatic cancer diagnosis 23 . Leu promoted adipose tissue protein synthesis through mTOR pathway 24 , and then adipocytes promoted ovarian cancer metastasis and provided energy for rapid tumor growth 25 . BCKDK promoted colorectal cancer and hepatocellular carcinoma metastasis and proliferation via the ERK signaling pathway 26-28 . Furthermore, BCKDK was highly expressed in DOX-induced ovarian cancer drug-resistant cell lines, and its expression level was twice as high as that of sensitive one 29 . Inhibition of BCKDK increased the sensitivity of ovarian cancer cells to paclitaxel 30 . We can't help but wonder if BCKDK could promote ovarian cancer proliferation and metastasis? Which pathways worked in this process?
In this study, We showed that the BCKDK had a higher expression in EOC patients versus normal tissues. The high expression of BCKDK was correlated with the advanced pathological grade for patients.
Overexpression of BCKDK increased the clone formation and migration ability of SKOV3 and OVCAR3 cells ex vivo. Knockdown of BCKDK inhibited EOC tumor progression ex vivo and in vivo. And we identi ed BCKDK as an upstream kinase of MEK, which up-regulated MEK/ERK signaling by interacting with MEK. The above results suggest that BCKDK may promote EOC proliferation and migration through enhancing the MEK/ERK signaling pathway.
CCGGCCAGCACCAGTTCCGTCATTCCTCGAGGAATGACGGAACTGGTGCTGGTTTTTG-3'. A mock shRNA with a sequence lacking signi cant homology to the human genome database was used as the mock shRNA. The sequence was: 5'-CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTG-3'. The sense and anti-sense oligonucleotides were synthesized, annealed and cloned into the pLKO.1-TRC cloning vector at the EcoRI and AgeI sites as described by the manufacturer 31 .

Western blot
Cells (0.8-2×10 6 ) were cultured in 10 cm diameter dishes to 70-80% con uence, and then starved without serum for 24h. Then the cells were treated with 40 ng/mL epidermal growth factor (EGF) (R&D catalog: 236-EG-200) for 15min. EGF is a well-known tumor promotion agent used to study malignant cell transformation in animal and cell models of cancer 32 . After this, cells were washed twice in PBS before being lysed in RIPA buffer (Coolaber, China). Then, samples were sonicated in 15 seconds intervals three times, and insoluble debris was removed by centrifugation at 13000 rpm for 15 min. Protein content was determined by BCA method (Coolaber, China). 30-120 µg of protein was separated by 10% SDS-PAGE and visualized by chemiluminescence (BIO-RAD, USA) in triplicate.
Growth curve analysis 2×10 5 cells were plated in each dish and counted at different times in triplicate, using a hemacytometer to generate a growth curve.
Anchorage-independent cell transformation assay Different cell lines (8×10 3 /well) were seeded in 6-well plates. The cells were then cultured in 1 mL of agar (Sigma-Aldrich Corp.) containing 10% FBS, 0.33% BME (Eagle basal medium, Sigma-Aldrich Corp.), 25 µg/mL gentamicin and 2 mM L-glutamine, with an additional 3ml of 2 mM L-glutamine, 0.5% BME agar containing 10% FBS and 25 µg/mL gentamicin being below. Then the cells were maintained in a 37°C, 5% CO 2 incubator for 7-14 days and the colonies were observed and assessed by microscopy in triplicate assays.

Tumor xenografts
Female athymic Balb/c nude mice (4-6-week-old) were purchased from Chongqing Tengxin Beer Experimental Animal Sales CO, LTD ( Chongqing, China). Mice were kept in speci c pathogen-free conditions according to the National Guidelines for Animal Usage in Research (set by the Chinese government) at the Chongqing Population and Family Planning Science and Technology Research Institute. Mice were divided and randomized into three groups. Each of the different cell lines (3×10 6 in 200 µl PBS) was injected subcutaneously into the right ank. The tumor volumes were measured every three day and were calculated with the formula: V = 0.52 (length ×width × height). Forty days after the injection, the mice were sacrifced with dislocation of cervical vertebra after injecting pentobarbital sodium (50mg/kg, i.p. ), 5min. The tumor tissues were prepared with para n sections after xation with formalin, and then stained with hematoxylin and eosin (H&E).
This study was approved by the ethical committee of Yichang Second People's Hospital. Samples were obtained with informed consent. Detailed information on patients was shown in Fig. 1d. The TMA was stained followed the standard protocol for IHC staining. High pressure repair was conducted with Tris/EDTA buffer (pH 10.0) for 120 ℃, 5 min. The sections were incubated with primary antibody against BCKDK (1:50). The DAB Detection Kit (Polymer) was used as the secondary detection system. Positive staining of brownish-yellow particles was located in the cytoplasm. The immuno-scores were calculated following the Remmele scoring method 33 , and the scores greater than 2 were used as positive group, the others were used as negative group. And the slides were independently examined by two pathologists.

Wound healing assay
The wound healing assays were applied to determine the migration ability of cells. 2×10 5 cells were cultured in a six-well plate until 80-90% con uence and then carefully scratched with a 10 µL pipette tip.
After washing three times with 1×PBS to remove detached cells, images in 10 different wound elds were captured at respective time points (0 h and 48 h) to evaluate the migration of cells in triplicate assays.
1× 10 5 cells suspended in 150 µL serum-free medium were seeded onto the upper chamber of 24-well plates, and 700 µL of medium with 10% FBS was added to the lower chamber. 48 h later, the medium was removed from the upper chamber. The non-migrating cells on the upper side of the chamber were removed thoroughly with a clean cotton swab. Then cells on the bottom side of the membrane were xed with 4% paraformaldehyde for 30 min, then stained with 0.1% of crystal violet (Sangon Biotech) for 15 min. Finally, the stained cells were counted by microscopy. Results represent the average number of cells in three elds per membrane in triplicate inserts. Immunoprecipitation HO8910-PM were seeded in 10cm dishes for 24h. Then, cells were harvested in IP buffer (150mM NaCl, 50mM tris-HCl pH 7.4, 1% NP40, 1mM DTT and 1mM EDTA). 2mg proteins were subjected to immunoprecipitation following the manufacturer's instructions. (Http://www.scbt.com/protocols.html? protocol=immunoprecipitation). The mouse source antibody was used for IP and the rabbit source antibody was used for western blotting.

Statistical analysis
All quantitative data in the present study were performed at least in triplicate. The results are expressed as the mean ± standard deviation. A two-tailed ANOVA or Student's t-test was used to evaluate the data.

Results
BCKDK is highly expressed in EOC and is associated with pathological grading of EOC patients BCKDK expression level was detected in 1 normal ovarian epithelial cell line and 5 EOC cell lines (Fig. 1a). The results showed that the BCKDK level of normal IOSE80 cells was the lowest. BCKDK was poorly expressed in SKOV3 and OVCAR3 cells, moderately expressed in HO8910 cells, and highly expressed in HO8910-PM and SW626 cells. Then, the expression level of BCKDK was also detected in EOC tissue and corresponding tumor adjacent tissue samples. The results demonstrated that expression level of BCKDK was higher in EOC tissue than corresponding adjacent tissue (Fig. 1b, 1c), and is associated with pathological grading of patients (Fig. 1e). And detailed information of patients is shown in Fig. 1d.
To test whether BCKDK can promote cell proliferation, BCKDK was overexpressed in SKOV3 and OVCAR3 cells which poorly expressed BCKDK. SKOV3 and OVCAR3 stable cell lines were generated with transfecting the pCMV-c-Flag or pCMV-BCKDK-Flag plasmid into cells, and the growth curves of SKOV3-Mock or SKOV3-BCKDK cells were compared. The results demonstrated that SKOV3-Mock cells grew slower than SKOV3-BCKDK cells (Fig. 2a, inner session indicating BCKDK overexpression). Then, the anchorage-independent growth of SKOV3-Mock or SKOV3-BCKDK was also compared, and the result showed that the number of colonies in SKOV3-Mock cell cultures was less than that in SKOV3-BCKDK cell cultures (Fig. 2c left panel). The corresponding statistical signi cance is indicated in the right panel of Fig. 2c. Similar results were observed in the cultures of OVCAR3-Mock or OVCAR3-BCKDK stable cells (Fig. 2b, 2d). These results indicated that BCKDK promoted EOC cell proliferation.

BCKDK promotes EOC cell migration
Since BCKDK is closely associated to tumor migration in colorectal caner 27 , we wondered whether BCKDK also regulated EOC migration. To test this hypothesis, wound healing assay and transwell assay were uesed to detect the effects of BCKDK on the migration of SKOV3 or OVCAR3 cells. The wound healing assay results were shown in Fig. 3a and 3b, which demonstrated that SKOV3/ OVCAR3-Mock cells had weaker wound healing ability than SKOV3/ OVCAR3-BCKDK cells. Furthermore, transwell assays results indicated that fewer cells migrated form upper chamber in SKOV3/ OVCAR3-Mock cells than in SKOV3/ OVCAR3-BCKDK cells. over-expression of BCKDK accelerate the migration of SKOV3 and OVCAR3 cells (Fig. 3c, 3d). Therefore, results above suggested that over-expression of BCKDK accelerated the migration capability of EOC cells.

Knockdown of BCKDK attenuates EOC tumor properties
To further verify the above hypothesis, BCKDK was knocked down in HO8910-PM EOC cells to generate the stable shMock cell lines and the stable shBCKDK cell lines (HO8910-PM-shMock, HO8910-PM-shBCKDK). The result in Fig. 4a inner session of left panel showed that BCKDK was knocked down by shRNA sequence for lines 2 and 4. And growth curves of HO8910-PM-shMock, or shBCKDK cell lines were tested. The results indicated that HO8910-PM-shMock cells grew dramatically faster than HO8910-PM-shBCKDK cells (Fig. 4a). Then, the anchorage-independent growth of the HO8910-PM-shMock or shBCKDK cell lines was analyzed, and the results demonstrated that the number of colonies in HO8910-PM-shMock cell cultures was much more than in HO8910-PM-shBCKDK cell cultures (Fig. 4b). And wound healing assay and transwell assay of the HO8910-PM-shMock or -shBCKDK cell lines were also analyzed, and the results suggested that knockdown of BCKDK attenuated the migration of HO8910-PM cells (Fig. 4c, 4d). Therefore, these above results indicated that knockdown of BCKDK in EOC cells inhibited tumorigenesis and migration ex vivo. Furthermore, tumor xenograft assays were also preformed in female athymic Balb/c nude mice. We injected HO8910-PM-shMock, or -shBCKDK cells (3×10 6 ) subcutaneously into the right ank, with tumor size assessed over 40 days. Tumors in HO8910-PM-shBCKDK-inoculated mice grew to a smaller size compared to those in HO8910-PM-shMock-inoculated mice (Fig. 5a, 5b). And the tumor growth curve was shown in Fig. 5c. The nal weight of tumor was shown in Fig. 5d. The tumor tissues dissected from these xenografts in the study were stained with hematoxylin & eosin (H&E) to verify these tissues belong to tumor tissues (Fig. 5e). These data indicated that inhibiting BCKDK expression in EOC cells signi cantly weaken their tumorigenic properties ex vivo and in vivo, and further veri ed that BCKDK promotes EOC cell proliferation and migration.

BCKDK interacts with MEK
As we con rmed that BCKDK directly interacted with MEK in colerectal cancer cells in before research 26 . We wondered whether BCKDK also interacted with MEK in EOC cells? Then, BCKDK was immunoprecipitated from HO8910-PM cells, and was detected with a MEK antibody by Western blotting. The results demonstrated that BCKDK could co-immunoprecipitate with MEK in HO8910-PM cells (Fig. 6e).
Taken together, our study indicates that BCKDK promotes EOC proliferation and migration by activating the MEK/ERK signaling pathway.

Discussion
OC is the deadliest gynecological tumor. The ve-year survival rate of OC patients is as low as 15-45% 34 . While, the survival rate of OC patients in FIGO's stage I can as high as 90% or above. To improve the diagnosis of ovarian cancer, a variety of serum markers have been developed and used in the diagnosis of ovarian cancer patients. In the 1980s, CA125 was reported as a tumor marker for ovarian tumors 35 .
However, tthe sensitivity and speci city of these markers are not as high as expected 36, 37 . Therefore, there is still urgently need to develop speci c serum markers that can be screened without internal inspections. As there are no external manifestations before the advanced stage of OC patients, and the fatality rate, including EOC, has not decreased signi cantly in the past 30 years 5, 38 . The new alternative or complementary targeted drugs also need to be researched and developed.
Previous studies demonstrated that the amino acid pro le could be an effective diagnostic tool in various cancer patients [39][40][41][42] , and some amino acids are associated with OC [43][44][45][46] , for example, leu 47 . The catabolism of BCAA is closely related to the development and progression of various tumors. Inhibition of BCAA catabolism can promote tumor growth and development 48,49 . Inhibiting the expression of BCAA catabolic enzymes can lead to the accumulation of BCAA in tumors, while the liver regeneration tissues were not accumulated 48 . Current research focuses on BCAT1 or BCAT2, which works in the rst step of BCAA catabolism, while there are relatively few studies on BCKDK, which is a key negative regulatory enzyme in BCAA catabolism [50][51][52][53] . Despite this, studies have shown that the overexpression of BCKDK promotes the growth and metastasis of various tumors 26-28 . In this study, we determined that BCKDK promoted the proliferation and metastasis of EOC, and BCKDK was expressed at higher levels in EOC tissues than in adjacent normal tissues (Fig. 1b) and is correlated with advanced pathological grade for patients (Fig. 1e). This suggests that BCKDK could be another potential biomarker for the treatment of EOC. Inhibitors targeting BCKDK will be examined in future research.
Current research of BCAA catabolism worked in tumors focused on BCAT. As the BCAT reaction is reversible and near equilibrium, its direction should respond to changes in concentrations of BCAA and BCKAs, and availability of the donors and acceptors of nitrogen, to some extent, the conclusion was opposite in different researches. For example, some studies con rmed that the high expression of BCAT promoted the transfer of the BCAA amino group to α-ketoglutarate (α-KG) to form glutamate and the corresponding branched-chain keto acids (BCKAs). The BCAA catabolism was increasing, then the BCA-CoA entering into tricarboxylic acid cycle that provided energy for tumor cells proliferation and growth 53,54 . The other studies veri ed that the catabolism of BCAA in tumor cells was decreasing, and the high expression of BCAT promoted the conversion of BCKAs to BCAA and α-KG, and then providing essential nutrients and energy for cancer growth [48][49]55 . Our research supports the second. The overexpression of BCKDK inhibits the conversion of BCKAs to BCA-CoA, which leads to the accumulation of BCKAs. Furthermore, the accumulation of BCKAs inhibits BCAA catabolism. Therefore, BCKAs are converted into BCAAs again through amination with the BCAT enzyme. In addition, there are studies showing that BCKDK and PPM1K make up a ChREBP-regulated node that integrates BCAA and lipid metabolism and promotes BCAAs as a material for fat synthesis for fat cells, which provide energy for tumor growth 56 . It has been proven that leu was increased in OC 47 . Other studies also found that the overexpression of BCAT promoted OC proliferation [51][52][53] . Therefore, our research gave a further understand of BCAA catabolism worked in the ovarian cancer. While, how BCKDK coordinated with BCAT to balance the BCAA metabolism? And whether they could directly regulate each other was still unclear.
Furthermore, to con rm the function of BCKDK in EOC, BCKDK was overexpressed in SKOV3 and OVCAR3 cells which poorly expressed BCKDK. BCKDK gain signi cantly promoted the proliferation and migration ex vivo, whereas knocked down the expression of BCKDK in HO8910-PM EOC cells reduced the proliferation and migration ex vivo and inhibited the tumor growth in vivo. Hence, these data supported the tumor-promoting function of BCKDK in EOC. These results were also consistent with previous ndings demonstrating that BCKDK is a key regulator of cell proliferation and metastasis in colorectal cancer and hepatocellular carcinoma 26-28 . Moreover, BCKDK promotes EOC proliferation and migration by activating the MEK/ERK signaling pathway. In agreement with our previous study, our previous study demonstrated that BCKDK promoted colorectal cancer proliferation by targetting the MEK1 26 . Another previous study also veri ed that BCKDK promoted hepatocellular cancer proliferation by MEK/ERK signaling pathway 28 . To our knowledge, this study was the rst to report the ectopic expression of BCKDK in EOC, and uncovered the mechanism that BCKDK regulates EOC proliferation and migration by MEK/ERK signaling pathway.
Other studies also showed that BCKDK was related to lipid metabolism, which was upregulated by APN 55 . In addition, BCKDK promoted tumor growth and metastasis by interacting with SRC or mTOR in colon cancer or hepatic carcinoma (Fig. 7) 27,28 . Therefore, in addition to the MEK-ERK pathway, whether the APN, SRC, or mTOR signaling pathways are also involved in this process still needs further examination. In addition, the drug resistance of ovarian cancer is a thorny issue, the mitochondria are closely related to apoptosis and autophagy-induced drug resistance 58-60 . As BCKDK is located in the mitochondria, and related to drug resistance in ovarian cancer [29][30] . What is the relevant mechanism? Many questions need to be addressed in the future. Due to the follow-up data was missing, overall survival rates and progressionfree survival rates were not analyzed in this study, future studies need to collect more clinical information.

Conclusions
These ndings indicated that the expression of BCKDK was upregulated in EOC tissues and that high expression of BCKDK was correlated with advanced pathological grade in patients. BCKDK promoted EOC proliferation and migration by activating MEK. Targeting the BCKDK-MEK axis may provide a new therapeutic strategy for treating patients with EOC.

Declarations
Ethics approval and consent to participate The study involving human tissues got approved by the Ethics Committee of Yichang Second People's Hospital. The study involving animals got approved by the Ethics Committee of the Chongqing Population and Family Planning Science and Technology Research Institute. (Ethic approval number: 2019D001). The study followed the tenets of the Declaration of Helsinki, and informed consent was obtained from each participant.

Consent for publication
All authors involved in the authorship are consent for publication in the current form.

Availability of data and materials
Not applicable.

Competing interests
The authors declare that they have no competing interests.