Introduction
Global incidence of breast cancer (BRCA) in women has increased significantly [
1], which has surpassed lung cancer and become the most commonly diagnosed cancer, with an estimated 2.3 million new cases (accounting for 11.7% of all cancer cases) in 2020; BRCA remained as the leading cause of female cancer-related death, accounting for 6.9% of mortalities worldwide [
2]. Despite advances in diagnosis and treatment, recurrence and death rates from BRCA remain high. Therefore, to improve overall survival and quality of life, it is urgent to discover new targets and to demonstrate molecular mechanisms for diagnosing and treating BRCA.
Energy metabolism in cancer has been extensively explored. Glycolysis, in particular, has become an attractive target for cancer therapy because many tumors exhibit a significant increase in glucose uptake compared with adjacent normal tissue [
3]. 3-Bromopyruvate, as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor, had exhibited anticancer efficacy in animal models [
4,
5]. Glycolysis involves a series of enzymes [
6], which may regulate downstream genes of mTOR signaling pathway. PKM2 activates mTORC1 signaling pathway by phosphorylating AKT1S1, which accelerates oncogenic growth in cancer cells [
7,
8]. The phosphoinositide 3-kinase (PI3K)/ AKT serine/threonine kinase (AKT) signaling pathway is involved in many cellular processes, including metabolism, metastasis [
9,
10] and epithelial–mesenchymal transformation (EMT) [
11]. Therefore, targeting glycolytic multienzyme system and related pathways are hypothesized be a novel therapeutic strategy in BRCA.
Triosephosphate isomerase 1 (TPI1) is regarded as a key enzyme in the glucose metabolism pathway and is involved in various biological functions, especially in metastatic phenotype [
12]. TPI1, also known as TIM, TPI, TPID, and HEL-S-49, is located in 12p13.31 [
13,
14]. TPI1 encodes a triosephosphate isomerase consisting of two identical subunits, which catalyzes the interconversion between glyceraldehyde-3-phosphate and dihydroxy-acetone phosphate in glycolysis and gluconeogenesis pathway, respectively [
15]. TPI1 has been proposed as a candidate oncogene, with its overexpression detected in several types of cancers, such as intrahepatic cholangiocarcinoma (ICC) [
16], gastric [
13], lung [
17,
18], and prostate cancer [
19]. In ICC, TPI1 was overexpressed and correlated with poor prognosis [
16]. TPI1 was recognized as a serum biomarker in patients with cancer, including BRCA [
20,
21], lung squamous cell carcinoma [
17] and hepatocellular carcinoma (HCC) [
22]. However, in HCC, TPI1 was identified as a tumor suppressor gene [
23]. These controversial findings indicate that exact roles of TPI1 in cancers, in particular BRCA, are yet to be identified.
In this study, we found that TPI1 is a marker of poor prognosis in BRCA. By promoting cell division cycle associated 5 (CDCA5) protein stabilization, TPI1 activates the PI3K/AKT/mTOR pathway, thereby enhancing metastasis and glycolysis. In addition, we also discovered that the ubiquitin-associated protein P62, interacts with TPI1 and promotes ubiquitin-dependent proteasome degradation of TPI1 in MDA-MB-231cells.Our study not only demonstrates, for the first time, the role of TPI1 in promoting breast cancer progression, but also provides a new strategy for targeted glycolysis therapy for breast cancer.
Materials and methods
Tissue specimens and patients
Totally 10 pairs of fresh breast tissues (paired BRCA tumor samples and matched adjacent normal tissue samples) were obtained from randomly selected patients for western blot (WB). We collected 28 normal samples and 362 BRCA samples from patients who were treated by surgical removal in 2007 for tissue microarray, with follow-up until July 2021.The overall survival (OS) and disease-free survival (DFS) were calculated as time from surgery until the occurrence of death and relapse, respectively. In addition, pathological sections from 50 BRCA patients were used for correlation analysis. All clinical samples were collected at Harbin Medical University Cancer Hospital and verified by histological and pathological examinations. This study was approved by the Ethics Committee of Harbin Medical University. All participating patients had provided written informed consent.
Immunohistochemistry (IHC)
The IHC assay was performed as previously described [
25]. Primary antibodies were listed in an Additional file
1: Table S1. The staining results were scored according to the following criteria: percentage of immunoreactive cells: 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%); and staining intensity: 0 (negative), 1 (weak), 2 (moderate), or 3 (intense). The final score for TPI1 expression was the multiplication of percentage and intensity. For statistical analysis, a final staining score of ≤ 7 was defined as low expression, whereas a score > 7 as high expression.
Cell culture, transfection and treatment
Human BRCA cell line and MCF-10A were secured from the Cancer Research Institute of Heilongjiang Province. 10% fetal bovine serum (ExCell Bio, Australia) and 1% penicillin/streptomycin (Solarbio, China) were added to all media. MCF7, T47D, UACC-812, and HCC70 cells were cultured in DMEM (Gibco, Life Technologies, California, USA), whereas MDA-MB-453 and SKBR-3 cells in RPMI 1640 (Gibco, California, USA), and MCF-10A cells in complete medium purchased from Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China [CAS: ZQ1311]). Cell lines were incubated in a humidified incubator at 37 °C with 5% CO2. MDA-MB-231 and MDA-MB-468 cells cultured in Leibovitz’s L-15 (PM151010, Procell, China) were placed in an incubator without CO2.
TPI1 knockdown (sh1, sh2, sh3) and vector (NC) by lentiviral system (Hanbio, Shanghai, China) with a puromycin selection marker were stably transfected into T47D cells. Furthermore, overexpressed TPI1 controlled with lentiviral system (Hanbio, Shanghai, China) was stably transfected into MDA-MB-231 cells according to the manufacturer’s instructions. Sequences of the interference TPI1 were listed in an Additional file
1: Table S2. Stable cells with lentiviral system were yielded after treatment with 1 μg/ml puromycin for 2 weeks. CDCA5 plasmid (CDCA5) and P62 plasmid (P62) were transfected into BRCA cells using jetPRIME® (Polyplus-transfection S.A, Illkirch, France) according to the manufacturer’s instructions, with empty vector plasmid as a negative control. All plasmids were purchased from Sino Biological (Beijing, China). Transfection efficiency was determined by qRT-PCR and WB, respectively. For cycloheximide (CHX) chase assay, cells were incubated with 200 μg/ml CHX (HY-12320, MCE, USA) for indicated durations (0, 2, 4, 6, 8, 10, and 12 h, respectively). LY294002 was used in cell-based assay (50 μM, 24 h) and mice models (75 mg/kg).
Western blot (WB) and qRT-PCR
WB was conducted as previously described [
25]. Briefly, cell lysates were obtained using RIPA lysis buffer (Beyotime, Shanghai, China). Concentrations of protein were confirmed by a BCA Protein Assay Kit (Thermo Scientific). Protein was separated using 10% or 12.5% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% milk, the membranes were incubated with primary antibody overnight at 4 °C. The next day, it was incubated for 1 h at room temperature with corresponding secondary antibodies and then was visualizing by an ECL Plus kit (Xin Saimei, China). The primary antibodies were listed in Additional file
1: Table S3.
Total RNA was extracted using TRNzol reagent (TIANGEN Biotech, Beijing, China). Complementary DNA was synthesized using a high-capacity cDNA reverse transcription kit. qRT-PCR was conducted with the Applied Biosystems 7500 Real-Time PCR System using a SYBR Green Real-Time PCR Master Mix kit (Takara, Dalian, China). Primers for qRT-PCR were listed in Additional file
1: Table S4.
Cell proliferation assays
For Cell Counting Kit-8 (CCK-8) assay, 5 × 103 cells/well were seeded in 96-well plates (Jet Biofil, Guangzhou) and cultured at 37 °C. 10 μl of CCK-8 (SEVEN BIOTECH, China) was mixed with 90 μl of medium. The mixture was added to each well at time points of 1, 2, 3, 4, and 5 days, respectively, after seeding, incubated for 2 h, and measured for absorbance at 450 nm.
For colony formation assay, cells (8 × 102/well) were seeded in 6-well plates, culture medium was changed every 2–3 days. Two weeks later, living cells were fixed, stained, and counted.
An Edu assay, a total of 5 × 103 cells/each well were seeded in 96-well plates for 2 days, added with Edu at a concentration of 50 μM, and cultured 2 h at 37 °C before fixation. This assay was carried out following the manufacturer’s protocol of Click-IT Edu Imaging Kits (Yuheng, Suzhou, China). Images were taken under an inverted fluorescence microscope.
Migration, invasion and wound healing assays
For migration assays, cells (3 × 104–7 × 104) were inserted in 200 μl of serum-free medium, and 600 μl medium with 10% FBS was added to the bottom chambers. After 24 h, cells were fixed in 4% paraformaldehyde and stained with crystal violet solution for 2 h. The number of cells per five randomly selected fields was counted under an inverted microscope (Leica, Germany). Then, cells were photographed and counted in five randomly selected fields.
For invasion assays, the upper basement membrane of the chamber was precoated with 30 μg Matrigel (356243, BD Biosciences) and cultured for 48 h at 37 °C. The procedure was the same as migration assay.
For wound healing assays, wounds were scratched on a monolayer cell by 10 μl pipette tips until 95% of the cells were covered in a 6-well plate. Then, wound healing images were taken at appropriate time points (24 h/48 h). The migration distance between the dotted lines was measured and normalized according to that of control cells.
Glucose uptake and lactate production measurement
Cells were cultured in 96-well plates for 48 h to examine glucose consumption rate and lactate production. The supernatants were collected for detection, measured for absorbance at 490 nm, and calculated according to the manufacturer’s instructions. The Glucose Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and Lactate Assay Kit (A019-2-1, Nanjing Jiancheng Bioengineering Institute) were applied for respective experiments.
In vivo assay
Female BALB/c nude mice (4–5 weeks old) were obtained from the Beijing Vital River Laboratory and housed under standard specific-pathogen-free conditions of the Animal Center of the Second Affiliated Hospital of Harbin Medical University.
The mice were randomly divided into three groups (n = 5/group). Two groups of mice were subcutaneously injected with MDA-MB-231 cells overexpressing TPI1-luciferase (5 × 106 cells in 200 µl phosphate buffered saline (PBS)/Matrigel [3:1]), while the remaining group was injected with MDA-MB-231-luciferase vector control cells. After primary tumor formation, five mice from TPI1 overexpressing group were randomly selected to receive LY294002 (75 mg/kg) intragastric administration 3 times a week for 3 weeks. T47D vector cells or shTPI1 cells (5 × 106 cells in 200 µl PBS/Matrigel [3:1]) were injected subcutaneously (n = 6/group). The tumor volume was monitored using vernier calipers every 4–5 days for a month, calculated by the formula (width2 × length)/2 (mm3). The mice were sacrificed after 35 days and tumor weight was measured. Tumor tissues were used for WB, H&E and IHC assays.
Immunoprecipitation (IP), silver staining and mass spectrometry assay
The protein A/G magnetic beads (MCE, HY-K0202) were incubated with antibodies at 4 °C for 1 h, followed by incubation with cell lysates based on the instructions. Subsequently, the beads were collected and subjected to immunoprecipitation (IP). A silver staining assay was performed according to the protocol provided by Beyotime Technology (P0017S, Beyotime, Shanghai, China). The antibody, magnetic beads and antigen were combined and boiled at 95 °C, and then the mass spectrometry results were obtained by Wuhan GeneCreate Biological Engineering. The antibodies used for the co-IP were TPI1 (Proteintech, 10713-1-AP), CDCA5 (Santa Cruz, sc-365319), and sequestosome-1/P62 (SQSTM1/P62) (Proteintech, 66184-1-Ig).
Immunofluorescence (IF) assays
The cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 for 10 min, and blocked by 10% normal goat serum for 30 min at room temperature. Cells were washed 3 times with PBS. Primary antibody was administered overnight at 4 °C. The next day, the cells were incubated with secondary antibody for 1 h and stained with DAPI (Beyotime, Shanghai, China) for 5 min at room temperature in a dark room. During each step, cells were washed 3 times with PBST. Then, pictures were taken under a confocal microscope (ZEISS LSM 800). The antibodies used were CDCA5 (Proteintech, 1:80 dilution, 67418-1-Ig), TPI1 (Proteintech, 1:50 dilution, 10713-1-AP), DyLight 649 Goat Anti-Rabbit IgG (A23620, Abbkine) or DyLight 488, and Goat Anti-Mouse IgG (A23210, Abbkine).
Ubiquitination assay
MDA-MB-231 cells transfected with Flag-Ub plasmid were pretreated with MG132 (10 μM) (MCE, New Jersey, USA) for 6 h and lysed. Then, co-IP was performed and analyzed by WB using an anti-ubiquitin antibody (1:100 dilution; ab134953, Abcam, Cambridge, MA, USA).
Statistical analysis
We performed statistical analyses using SPSS 22.0 software and GraphPad Prism 8.0 software, collecting data from no fewer than three independent experiments. The statistical results were expressed as mean ± standard deviation. The differences between two groups were analyzed using the student’s t-test and the chi-square test. Survival was calculated using the KM plotter method and the log-rank test. Spearman’s rank correlation coefficient (r) was used to quantify correlation analysis. A p value of < 0.05 (two-tailed) was considered statistically significant.
Discussion
In this study, TPI1 has been identified as a marker of poor prognosis in BRCA. By stabilizing CDCA5 protein, TPI1 activates PI3K/AKT/mTOR pathway, thereby enhancing tumor progression and glycolysis. In addition, P62 interacts with TPI1 and regulates protein stability. Our study, for the first time, demonstrates the roles of TPI1 in promoting breast cancer progression, and provides a new strategy for targeted glycolysis therapy.
High morbidity and mortality rates of breast cancer urge discovery for new effective therapy [
2,
26]. Dysregulated metabolism is a hallmark of cancer [
27]. Aerobic glycolysis is notable, in which cancer cells preferentially depend on aerobic glycolysis to obtain energy, even in the presence of abundant oxygen [
6]. Aerobic glycolysis promotes unrestricted growth and metastasis of cancer cells [
11]. Therefore, to target glycolysis is a promising therapeutic strategy for breast cancer. In the present study, TPI1 predicts poor prognosis in BRCA. Mechanistically, through stabilizing CDCA5, TPI1 activates PI3K/AKT/mTOR pathway, which in turn promotes breast cancer metastasis and glycolysis. Moreover, TPI1 undergoes ubiquitin-dependent proteasome degradation in the presence of P62 in BRCA cells (Fig.
8D).
In order to better understand regulatory mechanisms of aerobic glycolysis in BRCA, key genes were identified with KEGG. Multiple bioinformatics analyses identified TPI1 as a crucial glycolysis enzyme, which is overexpressed in BRCA and predicts poor prognosis. However, TPI1 was previously proposed as a tumor suppressor gene. In BRCA, TPI1 has been upregulated at cellular and tissue level. We noted that TPI1 was low expressed in triple negative breast cancer cell lines (HCC70 and MDA-MB-468). Somatic mutation frequency and gene expression level have a strong correlation in cancers [
28]. Just as BRCA1 mutation is more common in triple negative breast cancer, whereas BRCA2 mutations are more common in Luminal B breast cancer [
29]. Mutations at the dimer interface of TPI1 have been shown to lead to protein degradation and reduced TPI1 protein expression [
30]. TPI1
E104D is one of the common mutation sites of TPI1 [
31]. In addition, protein misfolding and accumulation of toxic substances may also contribute to reduced TPI1 expression [
32]. The expression of TPI1 was not associated with ER/PR and HER2 molecular typing related genes in our breast cancer tissue samples. So, we cannot conclude that TPI1 has different effects on different molecular subtypes of breast cancer. In our study, high expression of TPI1 is positively associated with clinical stages in 362 BRCA samples. Importantly, univariate and multivariate models indicate that TPI1 is an independent indicator for prognosis of BRCA patients. Thus, TPI1 may be a potential biomarker in BRCA.
TPI1 expression is higher in tissue and plasma of lung cancer patients than in cancer-free humans [
17]. Based on in vitro model and serological proteome, TPI1 may be assumed as a potential biomarker in lung carcinoma. On the contrary, TPI1 inhibits proliferation and metastasis via β-catenin and p53 signaling in HCC [
23]. However, no studies have explored if TPI1 links to oncogenic phenotypes in BRCA. For the first time, we demonstrate that TPI1 promotes proliferation, migration, invasion, and glycolysis both in vitro and in vivo. Thus, TPI spurs malignant phenotypes in BRCA cells.
Metastasis causes as much as 90% of cancer-associated mortality in general [
33‐
35], as a leading cause of BRCA deaths [
36]. EMT is a major mechanism leading to tumor invasion and metastasis [
37]. Meanwhile, PI3K/AKT/mTOR is a key pathway regulating proliferation, metastasis, and glycolysis [
38,
39]. Activation of AKT can activate pro-EMT transcriptional factors, directly or indirectly, to stimulate EMT process and induce pro-metastatic molecules, resulting in tumor metastasis [
40]. In addition, glycolytic enzyme PGK1 [
37] and enolase 1 (ENO1) [
11] promoted malignant phenotypes through AKT/mTOR pathway. Interestingly, upregulated TPI1 might activate PI3K/AKT/mTOR pathway based on GSEA. Meanwhile, expression of genes related to EMT and glucose metabolism were increased after overexpressing TPI1. Accordingly, knockdown TPI1 had the opposite effects. Treatment with LY294002 (PI3K pathway inhibitors) reversed phenotypes induced by TPI1 overexpression, demonstrating that TPI1 could regulate proliferation, EMT-related metastasis, and glucose metabolism through PI3K/AKT/mTOR pathway.
To elucidate how TPI1 regulates the PI3K/AKT/mTOR pathway, CDCA5 was identified as an important regulatory factor. CDCA5, also known as sororin, is a key player in sister chromatid cohesion and separation, and identified as a substrate of anaphase-promoting complex [
41,
42]. Multiple studies have revealed that CDCA5 is overexpressed in various cancers, such as lung cancer, oral squamous cell carcinoma, gastric cancer, and HCC. CDCA5 was overexpressed in bladder cancer tissues and activated PI3K/AKT/mTOR pathway [
41]. In HCC, CDCA5 promoted oncogenesis via AKT [
43]. Similarly, CDCA5 is an indicator of poor prognosis based on BRCA TCGA data. Moreover, overexpression of CDCA5 significantly enhances proliferation, migration, and glycolysis by activating PI3K/AKT/mTOR pathway. Subsequently, co-IP and IF analyses confirmed interaction between TPI1 and CDCA5 protein, as upregulated TPI1 stabilized CDCA5 protein by CHX assay. After CDCA5 plasmid was transferred into T47D/shTPI1 cells, proliferation, migration and glycolysis were strengthened. Furthermore, TPI1 activates PI3K/AKT/mTOR pathway by stabilizing CDCA5 expression. Consistently, Ser209 phosphorylation on CDCA5 protein by ERK promoted growth or survival of lung cancer cells [
44]. However, how TPI1 affects CDCA5 is still unclear, which should be elucidated in future studies.
Notably p62 was identified as a direct regulator of TPI1 by silver staining and mass spectrometry. The interaction between TPI1 and p62 was verified by co-IP assay. P62 contains several structural motifs, which may participate in forming multimeric signaling complexes. The C-terminal ubiquitin-associated domain of P62 binds to ubiquitin-conjugated proteins [
45], while its N-terminal PB domain targets proteasome. Thus, P62 can act as a cargo receptor to promote proteasome degradation of ubiquitin proteins [
46]. P62 promoted the ubiquitination of Tensin-2 through PB1 domain, leading to proteasome mediated degradation [
47]. P62 also induced the ubiquitination and degradation of glycolytic enzyme 6-phosphofructo-2-kinase [
34,
46]. Consistent with our results, overexpression of P62 decreased the protein expression of TPI1 through ubiquitination degradation. P62 may participate in the proteasome degradation of TPI1 as a cargo receptor. P62 is highly expressed in breast cancer. However, in our study, TPI1 is overexpressed in breast cancer, while p62 promotes degradation of TPI1. P62 [
34] and TPI1 are expressed differently in different molecular types of breast cancer. The expression of P62 was relatively high in MDA-MB-231 cell [
48], while the expression of TPI1 was relatively low. Thus, P62 induced ubiquitination of TPI1 in MDA-MB-231 cells. However, which fragment of P62 mediates its interaction with TPI1, and whether this phenomenon occurs in different molecular types remain to be explored. In short, we have only reported preliminary results on the relationship between P62 and TPI1 in this article, and we will continue in-depth studies in the future.
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