TSC22D2 interacts with PKM2 and inhibits cell growth in colorectal cancer

  • Authors:
    • Fang Liang
    • Qiao Li
    • Xiayu Li
    • Zheng Li
    • Zhaojian Gong
    • Hao Deng
    • Bo Xiang
    • Ming Zhou
    • Xiaoling Li
    • Guiyuan Li
    • Zhaoyang Zeng
    • Wei Xiong
  • View Affiliations

  • Published online on: July 4, 2016     https://doi.org/10.3892/ijo.2016.3599
  • Pages: 1046-1056
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

We previously identified TSC22D2 (transforming growth factor β-stimulated clone 22 domain family, member 2) as a novel cancer-associated gene in a rare multi-cancer family. However, its role in tumor development remains completely unknown. In this study, we found that TSC22D2 was significantly downregulated in colorectal cancer (CRC) and that TSC22D2 overexpression inhibited cell growth. Using a co-immunoprecipitation (co-IP) assay combined with mass spectrometry analysis to identify TSC22D2-interacting proteins, we demonstrated that TSC22D2 interacts with pyruvate kinase isoform M2 (PKM2). These findings were confirmed by the results of immunoprecipitation and immunofluorescence assays. Moreover, overexpression of TSC22D2 reduced the level of nuclear PKM2 and suppressed cyclin D1 expression. Collectively, our study reveals a growth suppressor function of TSC22D2 that is at least partially dependent on the TSC22D2-PKM2-cyclinD1 regulatory axis. In addition, our data provide important clues that might contribute to future studies evaluating the role of TSC22D2.

Introduction

In a previous study, we performed a genome-wide linkage analysis of 24 core family members in a rare multi-cancer family. The family included 103 members spanning six generations, and 15 members had been diagnosed with various tumor types, including colorectal cancer (CRC), breast cancer, endometrial carcinoma and gastric cancer. The disease susceptibility locus in this family was mapped to chromosome 3q24-26. A novel mutant in the TSC22D2 gene located in chromosome band 3q24-26 co-segregated with the cancer phenotype, as demonstrated by exome sequencing (1).

TSC22D2 is a member of the TSC-22 domain family comprising putative transcription factors that are characterized by a carboxy-terminal leucine zipper and an adjacent TSC-box. However, there are few studies describing TSC22D2, and its role in tumor development remains largely unknown.

Accumulating evidence shows that many TSC22D2 family proteins interact with other proteins to form macromolecular complexes and are involved in a broad range of biological processes. For instance, TSC22D1 (referred as TSC-22) can suppress tumor growth by binding to p53 (2) and can enhance TGF-β signaling by associating with Smad4 (3); TSC22D3 (usually called GILZ) plays multiple biological functions dependent on its interaction with other proteins, including NF-κB, Ras and p53 (46). Accordingly, the identification of the TSC22D2-associated proteins or macromolecular complexes is important to precisely understand the underlying mechanism of TSC22D2 in human carcinogenesis.

In this study, we investigated the role of TSC22D2 in colorectal cancer, the most common malignancy in the multi-cancer family. We found TSC22D2 was significantly downregulated in CRC. Further functional study showed that TSC22D2 overexpression can inhibit CRC cell growth. To precisely define the underlying mechanism by which TSC22D2 influenced CRC cell growth, we used co-immunoprecipitation (co-IP) and mass spectrometry approaches to identify the proteins or macromolecular complexes that interact with TSC22D2, and gained 142 candidate TSC22D2-binding proteins that were associated with a variety of cellular processes. Moreover, we determined that TSC22D2 physically associates with pyruvate kinase isoform M2 (PKM2), a glycolytic enzyme reported to be associated with the growth and survival of multiple cancer cell types (79), and demonstrated that TSC22D2 overexpression reduces nuclear PKM2 levels and represses the expression of cyclin D1 (a downstream target gene of nuclear PKM2).

Materials and methods

CRC samples

Surgical cancer tissue specimens and adjacent normal mucosa (≥5 cm away from the tumor margins) were obtained from 14 patients with colorectal cancer who had undergone surgery at the Third Xiangya Hospital of Central South University. Informed written consent was obtained from each patient, and the research protocols were approved by the Medical Ethics Committee of Xiangya Medical College. No patients had received preoperative adjuvant therapy. After collection, all tissue samples were immediately frozen in liquid nitrogen until use.

Plasmid construction

The full length TSC22D2 gene was cloned into the pIRESneo3-Flag vector with NheI and StuI (Takara, Dalian, China) sites. The TSC22D2 gene was amplified using the following primers: TSC22D2 sense primer (5′-ACG TGCTAGCGCCACCATGTCCAAGATGCCGGCCAA-3′), TSC22D2 anti-sense primer (5′-ACTGAGGCCTTTATGCTG AGGAGACATTCG-3′). Full-length PKM2 was generously provided by Professor Xianghuo He (Shanghai Medical College, Fudan University, Shanghai, China) and has been previously described (10).

Cell lines and cell culture

The SW480 (human colorectal carcinoma) and HEK293 (human embryonic kidney) cell lines were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. The HeLa human cervical carcinoma cells were grown in 1640 medium (Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin.

Cells were transfected using the Lipofectamine 3000 reagent (Life Technologies) according to the manufacturer’s instructions. HEK293, SW480 and HeLa cells stably expressing Flag-tagged TSC22D2 (Flag-TSC22D2) or the control vector (Flag-NC) were obtained by puromycin (600–1,000 ng/ml) (Life Technologies) selection for one month. All cells were maintained under standard culture conditions (37°C, 5% CO2).

RNA isolation and quantitative real-time PCR

Total RNAs were isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and were converted to cDNA using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. All real-time PCR reactions were performed with SYBR Green (Takara) using the Bio-Rad CFX Connect Real-Time system (Bio-Rad, Hercules, CA, USA). The following PCR program was used: denaturation at 95°C for 30 sec, followed by 40 cycles consisting of denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. A melting curve analysis was applied to assess the specificity of the amplified PCR products. The amount of each target gene was quantified by the comparative CT method using GAPDH as the normalization control. The following primers were synthesized from Life Technologies and used to amplify TSC22D2, CCND1 and GAPDH: TSC22D2 forward primer (5′-TGAT GGTGATGAAGACAGTGC-3′), TSC22D2 reverse primer (5′-GGGTTGGGAGTTGGGATAAT-3′); CCND1 forward primer (5′-CAACCTCCTCAACGACC-3′), CCND1 reverse primer (5′-CTTCTGTTCCTCGCAGAC-3′); GAPDH forward primer (5′-AACGGATTTGGTCGTATTGG-3′), GAPDH reverse primer (5′-TTGATTTTGGAGGGATCTCG-3′). All experiments were carried out in triplicate.

Total protein, subcellular fractionation and western blot analysis

Nuclear and cytosolic extracts were fractionated using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Bremen, Germany) according to the manufacturer’s protocol. To isolate total protein, cells were harvested by scraping and transferred to SDS sample buffer supplemented with a protease inhibitor cocktail and the PhosSTOP phosphatase inhibitor (Roche, Pleasanton, CA, USA). Equal levels of protein from the samples were separated using SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Millipore Corp., Billerica, MA, USA). The membrane was blocked with PBST containing 5% skim milk for 1 h and then incubated overnight with the indicated primary antibodies at 4°C. The membrane was washed three times in PBST and subsequently incubated with an HRP-conjugated secondary antibody for 2 h at 37°C. The membranes were stripped of the primary antibodies and re-probed with additional antibodies as necessary. Bound antibodies were visualized using the enhanced chemiluminescence kit (Millipore).

The following antibodies were used in this study: monoclonal mouse anti-Flag (F1804, Sigma, St. Louis, MO, USA), anti-HA (H3663, Sigma), polyclonal rabbit anti-human TSC22D2 (AV39137, Sigma), polyclonal rabbit anti-human PKM2 (Cell Signaling, Danvers, MA, USA) and anti-CCND1 (Cell Signaling). An antibody against GAPDH (Cell Signaling) served as an endogenous control for equal loading, and anti-H3 (Beyotime, China) served as a nuclear control.

Cell proliferation assays

Cell proliferation was measured by counting viable cells using the Z2 Particle Counter and Size Analyzer Beckman Coulter (Miami, FL, USA). Briefly, SW480 and HeLa cells stably expressing Flag-tagged TSC22D2 (Flag-TSC22D2) or the control vector (Flag-NC) were seeded at 1.2×104 cells per well in 24-well plates in quadruplicate, the number of viable cells in each well was measured at 0, 1, 2, 3, 4 and 5 days.

Colony formation assays

For the colony formation assays, SW480 and HeLa cells were seeded in a 6-well plate at a density of 500 and 200 cells per well respectively, and incubated at 37°C with 5% CO2 for 2 weeks. The medium was changed every 3–4 days. At the end of the incubation, the medium was removed, and the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, stained with crystal violet for 30 min at room temperature, washed and imaged. Colonies of >50 cells were identified using an inverted microscope (Olympus IX50; Olympus Corp., Tokyo, Japan) and counted.

Flow cytometry cell cycle assays

SW480 and HeLa stable cells were plated at a density of 4×105 cells per well in 6-well plates and cultured in medium without serum (starvation treatment) for 12 h to synchronize cell cycle progression. Cells were incubated in serum-containing growth medium for an additional 24–36 h and subsequently trypsinized, washed, fixed with 70% ice cold ethanol overnight at 4°C. DNA was stained by incubating the cells in PBS containing PI (50 μg/ml) and RNase A (50 μg/ml). The cell cycle distribution was determined by flow cytometric analysis using a MoFlo™ XDP High-Performance Cell Sorter (Beckman Coulter, Brea, CA, USA) and the data were analyzed using Summit v.5.2 software.

Co-immunoprecipitation (co-IP) assays

HEK293 Flag-NC, HEK293 Flag-TSC22D2, SW480 Flag-NC, SW480 Flag-TSC22D2 stably transfected cells and HEK293T cells transiently cotransfected with Flag-TSC22D2 and HA-PKM2 were seeded in 100-mm dishes. The cells were lysed in modified lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCI, 50 mM NaF, 1 mM Na3VO4, 30 mM sodium pyrophosphate, 0.5% NP-40 and 0.5 mM PMSF (Sigma) supplemented with an EDTA-free protease inhibitor cocktail (Roche). Lysates were incubated with 5 μg of primary antibody overnight at 4°C. Then, 40 μl of a 1:1 slurry of protein A/G Plus-agarose (Santa Cruz, CA, USA) and a specific antibody were added to the cells for ≥4 h at 4°C. The immunoprecipitates were washed four times with lysis buffer and boiled with sample loading buffer and then analyzed by western blotting.

Mass spectrometry

Proteins were resolved on a 10% polyacrylamide gel, stained with Coomassie Brilliant Blue (R250) and subjected to mass spectrometry. Briefly, the stained SDS-PAGE gels were scanned, and stained bands were excised, cut into small (<1 mm3) pieces and washed three times by repetitive dehydration and hydration with acetonitrile and ammonium bicarbonate, respectively. Proteins were in-gel reduced in the presence of 25 mM DTT and 25 mM NH4HCO3 for 1 h at 56°C, immediately alkylated using 50 mM IAA and 25 mM NH4HCO3 for 30 min at room temperature in the dark and digested overnight at 37°C with 5 μg of trypsin in 25 mM NH4HCO3. Digested peptides were recovered, dried and resuspended in 50% CAN and 0.1% TFA. The peptide mixture was analyzed by nano-liquid chromatography-tandem mass spectrometry using an LTQ Velos-Orbitrap MS (Thermo Scientific, Waltham, MA, USA) coupled to an Ultimate RSLC nano-LC system (Dionex). Briefly, Raw MS data files were processed using the Proteome Discoverer v.1.4 (Thermo Scientific). Processed files were searched against the Swiss-Prot human database using the Sequest HT search engine. Mass tolerances for precursor and fragment ions were set to 10 ppm and 0.8 Da, respectively, in the database searches. Peptide identification with false discovery rates <1% (q-value <0.01) were discarded.

Immunofluorescence

TSC22D2, HEK293, SW480 and HeLa cells grown on culture slides were maintained in a 35-mm dish. After cultured for 24 h, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.25% Triton X-100 for 30 min and blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature. Fixed cells were then incubated with rabbit anti-human TSC22D2 antibody (1:1,000) at 4°C overnight, washed three times in PBS and stained with Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, 1:1,000) for 2 h in the dark at room temperature. The cells were then incubated with DAPI to stain the nuclei and visualized using a confocal laser scanning microscope (Olympus Corp.).

For the colocalization assays, HEK293T transfected with Flag-TSC22D2 and HA-PKM2 were incubated with the anti-Flag mouse monoclonal antibody (1:1,000) and the anti-PKM2 rabbit monoclonal antibody (1:1,000) at 4°C overnight and subsequently stained with Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen, 1:1,000) and Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen, 1:1,000) for 2 h at room temperature. After incubation with DAPI to stain the nuclei, the cells were visualized using a confocal laser scanning microscope (UltraView Vox; Perkin-Elmer, Waltham, MA, USA).

Statistical analysis

The experiments were repeated at least three times. All data are presented as the mean ± standard deviation. Paired Student’s t-test (two-tailed) and unpaired Student’s t-test (two-tailed) were performed to compare the means of two samples unless otherwise indicated. A p-value <0.05 was considered statistically significant. All statistical analyses were performed using the GraphPad Prism software (Graphpad Software, Inc.).

Results

Expression of TSC22D2 is decreased in colorectal cancer samples

We focused on human colorectal cancer in this study because it was the most frequent malignancy in the multi-cancer family. We first evaluated the expression of TSC22D2 transcripts in colorectal cancer by interrogating the public gene expression GEO databases (GSE8671, GSE32323 and GSE24514) and found that the expression of TSC22D2 was lower in human colorectal cancer samples than in non-tumor samples (Fig. 1A). To confirm these observations, the expression of TSC22D2 was evaluated in 14 pairs of human CRC samples (primary tumor tissues and paired adjacent non-tumor tissues). TSC22D2 expression was reduced in 13 of 14 (92.9%) of colorectal tumors compared with the paired adjacent non-tumor tissues (Fig. 1B, p=0.015), consistent with the data provided by the GEO database. These results suggest that TSC22D2 plays a suppressor role in CRC tumorigenesis.

TSC22D2 suppresses CRC cell growth

To investigate the function of TSC22D2 in colorectal cancer, the full-length TSC22D2 gene tagged with three Flag tags was cloned into the pIRESneo3 plasmid and stably transfected into SW480 and HeLa cells. The expression of TSC22D2 was confirmed by qRT-PCR and western blot analysis (Fig. 2A). Immunofluorescence assays demonstrated that TSC22D2 predominantly localized to the cytoplasm under steady state conditions (Fig. 2B). Cell proliferation assays and clone formation assays were conducted to investigate the effect of TSC22D2 on cell proliferation. TSC22D2-overexpressing cells exhibited a significantly slower growth rate (Fig. 3A) and a reduced number of clones (Fig. 3B) compared with control cells, indicating that TSC22D2 functions as a suppressor in CRC cell growth. Furthermore, flow cytometry (FCM) revealed that TSC22D2 induced a substantial increase in the proportion of cells at the G0/G1 phase and a concomitant decrease in the proportion of cells at the S phase of the cell cycle (Fig. 3C). These results indicate that TSC22D2 induces cell cycle arrest at the G0/G1 phase.

Identification of TSC22D2 binding partners

To explore the underlying mechanism by which TSC22D2 exerts its tumor-suppressive function, we performed co-IP assays (Fig. 4A) and LC-MS/MS analysis (Fig. 4B) in SW480 and HEK293 cells stably transfected with TSC22D2. We identified 90 and 79 candidate proteins in SW480 and HEK293 samples, respectively (Fig. 4C). There were 27 overlapping results for proteins associated with metabolism (PKM2, ATP5A1, LDH and ENO1), stress response and inflammation (STK39, HSPD1, HSP90AA1 and HSP90AB1), cell proliferation (NRBP1, PKM2, YWHAQ, LDHB, PCNA and PHB2), apoptosis (HSPD1, CAPN1, DNAJA1, HSP90AA1 and RPS3) and migration/metastasis (YWHAQ, HSPD1 and PHB2) between the 2 cell lines (Fig. 4D and Table I). These findings suggest that TSC22D2 plays a role in these biological pathways in cancer cells.

Table I

TSC22D2-interacting proteins identified in both SW480 and HEK293 cells.

Table I

TSC22D2-interacting proteins identified in both SW480 and HEK293 cells.

Gene symbolDescriptionConfirmed role in cancer
TSC22D2Fesponse to osmotic stress
NRBP1Subcellular trafficking between the endoplasmic reticulum and Golgi apparatusPotentially plays a suppressive role in tumor progression
TSC22D4Sequence-specific DNA binding transcription factor activity
TUFMProtein translation in mitochondria and oxidative phosphorylation; regulates type I interferon and autophagy
PKM2Involved in glycolysis; transcription factor activityImportant for tumor cell proliferation and survival
ATP5A1A subunit of mitochondrial ATP synthase; involved in energy metabolism
STK39A serine/threonine kinase that might function in the cellular stress response pathway
YWHAQBelongs to the 14-3-3 family of proteins, which mediate signal transduction by binding to phosphoserine-containing proteinsCoordinates the regulation of proliferation, survival and metastasis
HSPD1Putative signaling molecule in the innate immune system; implicated in mitochondrial protein import and macromolecular assemblyRegulates tumor cell apoptosis, survival and metastasis
LDHBCatalyzes the interconversion of pyruvate and lactate and the concomitant interconversion of NADH and NAD+ in a post-glycolysis processA metabolic marker in cancer and is potentially associated with cell growth
PCNACofactor of DNA polymerase delta; DNA damage responseImportant for cancer cell proliferation
PHB2Transcriptional repression; likely to be involved in regulating mitochondrial respiration activity and aging; associated with the cell cycleRequired for cancer cell proliferation, and potentially regulates cell migration
EIF4A1RNA helicase; translation initiation factor activity; participates in the TGF-β pathway and p70S6K signaling
ENO1Functions as a glycolytic enzyme and a transcriptional repressorPromotes cell proliferation and possesses oncogenic activity
FLNAA well-characterized actin cross-linking protein; anchors various transmembrane proteins to the actin cytoskeleton and serves as a scaffold for a wide range of cytoplasmic signaling proteinsHas a tumor-promoting effect when localized to the cytoplasm, and suppresses tumor growth and inhibits metastasis when localized to the nucleus
CAPN1Catalyzes limited proteolysis; involved in ERK signaling and apoptosisParticipates in apoptosis
CCT3Molecular chaperone; assists in the folding of proteins stimulated by ATP hydrolysis
DNAJA1Positive regulation of viral replication; plays a role in protein transport into mitochondria via its role as a co-chaperone; protects cells from apoptosisSuppresses the anti-apoptosis state in cancer
EEF1GTranslation elongation factor activity; likely to play a role in anchoring the translation complex to other cellular components
EEF2Promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome; an essential factor for protein synthesisFunctions as an oncogene in cancer cell growth
HNRNPMAssociated with pre-mRNAs in the nucleus and appears to influence pre-mRNA processing and other aspects of mRNA metabolism and transportRegulates cell cycle progression, promotes cell growth and invasion; promotes TGFβ-induced EMT and metastasis
HSP90AA1
HSP90AB1
Mediates LPS-induced inflammatory response; molecular chaperone that promotes the maturation, structural maintenance and proper regulation of specific target proteins involved in multiple processes, including cell cycle control and signal transductionIs involved in cell apoptosis and chemosensitivity
Methylated HSP90AB1 accelerates cancer cell proliferation
NUP62Forms a gateway that regulates the flow of macromolecules between the nucleus and the cytoplasm (involved in nuclear-cytoplasmic transport)Nup62 knockdown reduces cancer cell growth and viability
RPS3A component of the 40S small ribosomal subunit; plays a role in the repair of damaged DNARegulates cell growth, apoptosis and GLI2-mediated migration and invasion
SERPINH1A collagen-specific molecular chaperone that plays a role in collagen biosynthesisEnhances cell growth, migration and invasion
TUBB2AKey participant in various processes, including mitosis and intracellular transport
TSC22D2 physically associates with PKM2

We selected PKM2, a glycolytic enzyme that catalyzes the conversion of phosphoenopyruvate (PEP) and ADP to pyruvate and ATP for further analysis due to its relatively high mass spectrometry score and its previously reported role in cancer cell growth and survival (79).

The interaction between PKM2 and TSC22D2 was further confirmed by IP analysis of HEK293T cells transfected with HA-tagged PKM2 and Flag-tagged TSC22D2 (Fig. 5A). To determine whether TSC22D2 and PKM2 co-localize in cells, we performed immunofluorescence staining with anti-Flag and anti-PKM2 antibodies in HEK293 cells. The results revealed that TSC22D2 and PKM2 co-localized primarily in the cytoplasm (Pearson’s correlation, 0.954) (Fig. 5B). Taken together, these data suggest that TSC22D2 physically associates with PKM2.

TSC22D2 reduces the level of nuclear PKM2 and suppresses the expression of cyclin D1

Accumulating evidence indicates that PKM2 is crucial for aerobic glycolysis and that it provides a growth advantage to tumors (11,12). To determine if the effect of TSC22D2 on cell growth is mediated by PKM2, we evaluated the expression of PKM2 and observed that there were no significant changes in PKM2 expression at the mRNA and protein level in cells overexpressing TSC22D2 (Fig. 6A).

PKM2 predominantly localizes to the cytosol and plays an important role in metabolic functions. Recently, several independent studies demonstrated that PKM2 can translocate to the nucleus and induce the expression of gene products required for tumorigenesis by activating multiple transcription factors (1318). Thus, we asked whether TSC22D2 could affect the nuclear function of PKM2. A subcellular fractionation analysis was performed, and a slight but significant decrease in the nuclear levels of PKM2 was observed in TSC22D2-overexpressing cells compared with the control cells (Fig. 6B).

Cyclin D1, a key regulator required for the G1/S cell cycle transition, was reported to be a downstream gene of nuclear PKM2 (15,16). Therefore, we next investigated the effect of TSC22D2 on cyclin D1 expression. Intriguingly, TSC22D2 repressed the expression of cyclin D1 at both mRNA and protein levels (Fig. 6C and D), suggesting that TSC22D2 might inhibit cell growth by the influence on nuclear PKM2 and cyclin D1.

Discussion

We previously identified a novel cancer-associated gene, TSC22D2, by performing genome-wide linkage analysis and exome sequencing in samples derived from a multi-cancer family. TSC22D2 is a member of the TSC-22 domain family of proteins that are characterized by a carboxy-terminal leucine zipper and an adjacent TSC-box. However, its role in tumorigenesis remains largely unclear. In this study, we demonstrated that TSC22D2 is expressed at low levels in CRC and that TSC22D2 overexpression significantly inhibited the growth rate of cancer cells. Taken together, these results suggest that TSC22D2 might play a suppressive role in tumorigenesis.

Members of the TSC22 domain family are reported to interact with other proteins to form macromolecular complexes associated with a broad range of biological processes. TSC22D1 (also referred as TSC-22) binds to p53 and protects it from poly-ubiquitination-mediated degradation (2), it can enhance TGF-β signaling by associating with Smad4 (3). TSC22D3 (commonly referred to as GILZ) exhibits multiple biological functions that are dependent on its interaction with other proteins, including NF-κB, Ras and p53 (46). TSC22D4 is capable of heterodimerizing with apoptosis-inducing factor (AIF) and might participate in apoptosis (19). However, proteins that interact with TSC22D2 have not been identified.

In this study, we conducted co-IP assays combined with LC-MS/MS approaches to identify TSC22D2-interacting proteins. The results were enriched for proteins associated with the regulation of gene transcription, suggesting that TSC22D2 plays a role in transcription. TSC22 domain proteins have a classic leucine zipper and a TSC-Box structure. The leucine zipper might be a characteristic of a novel category of DNA binding proteins (2022) and the TSC-box is essential for nuclear localization and transcriptional activity (23). Based on these observations, TSC22 domain proteins are considered to be putative transcription factors. They primarily localize to the cytoplasm, but in response to specific stimuli, they can also translocate to the nucleus. Previous studies have demonstrated that TSC22D1 can translocate to the nucleus and suppress cell division in response to anti-proliferative stimuli (24,25). TSC22D1 and TSC22D3 transcriptional activity has been previously reported, and the transcriptional activity of TSC22D2 has yet to be further confirmed.

Our analysis revealed that TSC22D2 interacts with multiple proteins associated with metabolism (PKM2, ATP5A1, LDHB and ENO1), the stress response and inflammation (STK39, HSPD1, HSP90AA1 and HSP90AB1), cell proliferation (NRBP1, PKM2, YWHAQ, LDHB, PCNA and PHB2), apoptosis (HSPD1, CAPN1, DNAJA1, HSP90AA1 and RPS3) and migration/metastasis (YWHAQ, HSPD1 and PHB2) (Table I), implying that the function of TSC22D2 in tumor cells might be mediated by these processes. Moreover, the interaction between NRBP1 and TSC22D2 was inadvertently previously reported (26). In addition, TSC22 domain proteins have been shown to homodimerize and heterodimerize with other family members (27). In this study, western blotting and mass spectrometry analyses revealed that TSC22D2 primarily exists as a dimer, but that it might also heterodimerize with TSC22D4.

Given that TSC22D2 was capable of inhibiting the growth of cancer cells, we focused on the PKM2 gene, which was previously reported to be required for tumor growth (79). Pyruvate kinase M2 (PKM2) is a pyruvate kinase that regulates the final rate-limiting step of glycolysis by catalyzing the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP to generate pyruvate and ATP. PKM2 exhibits active pyruvate kinase activity as well as protein kinase activity that is primarily associated with transcriptional regulation (reviewed in refs. 11,12). Elevated PKM2 expression is currently considered to be a common characteristic of cancers.

PKM2 primarily localized to the cytoplasm. In addition to its well characterized role in glycolysis, PKM2 can translocate to the nucleus to stimulate the activity of multiple transcription factors, including HIF-1, STAT3, β-catenin and Oct-4, thereby increasing the expression of gene products that are required for tumor growth (1618,28). For example, activation of EGFR in human glioblastoma cells induces the nuclear translocation of PKM2. In addition, the interaction between PKM2 and β-catenin transactivates factors that induce HDAC3 removal from the CCND1 promoter, histone H3 acetylation and cyclin D1 expression (16). An increasing body of evidence indicates that the nuclear translocation of PKM2 promotes the Warburg effect and tumorigenesis (1318). In this study, we found that TSC22D2 did not regulate the total PKM2 protein levels, but that it decreased the level of nuclear PKM2. Although some studies have suggested that PKM2-interacting proteins impair PKM2 nuclear translocation by altering the balance between its monomer, dimer and tetramer forms or by mediating epigenetic modifications (14,29), further studies are required to confirm the mechanism by which TSC22D2 regulates the nuclear PKM2 level.

In conclusion, this is the first study to report the tumor suppressor role of TSC22D2 in cancer and to identify putative TSC22D2-interacting proteins using interactome analysis. The candidate interactions that we identified provide important clues that will facilitate future studies exploring TSC22D2 functions. The interaction between TSC22D2 and PKM2 may partially account for the growth suppressor function of TSC22D2. It is important to note that further studies of TSC22D2 complexes are required for gaining additional insight into the functions and precise mechanisms associated with TSC22D2 in cancer.

Acknowledgements

We are grateful for the gifts of the HA-PKM2 plasmids from Professor Xianghuo He. We also would like to thank the High Resolution Mass Spectrometry Laboratory of Advanced Research Center in Central South University for the technological assistance in proteomic examinations. This study was supported in part by grants from The National Natural Science Foundation of China (81272298, 81372907, 81301757, 81472531, 81402009, 81572787 and 81528019), the Natural Science Foundation of Hunan Province (14JJ1010 and 2015JJ1022) and the project from Central South University (2013zzts071).

Abbreviations:

TSC22D2

TSC22 domain family member 2

PKM2

pyruvate kinase isoform M2

CRC

colorectal cancer

References

1 

Li Q, Chen P, Zeng Z, Liang F, Song Y, Xiong F, Li X, Gong Z, Zhou M, Xiang B, et al: Yeast two-hybrid screening identified WDR77 as a novel interacting partner of TSC22D2. Tumor Biol. (In press).

2 

Yoon CH, Rho SB, Kim ST, Kho S, Park J, Jang IS, Woo S, Kim SS, Lee JH and Lee SH: Crucial role of TSC-22 in preventing the proteasomal degradation of p53 in cervical cancer. PLoS One. 7:e420062012. View Article : Google Scholar : PubMed/NCBI

3 

Choi SJ, Moon JH, Ahn YW, Ahn JH, Kim DU and Han TH: Tsc-22 enhances TGF-beta signaling by associating with Smad4 and induces erythroid cell differentiation. Mol Cell Biochem. 271:23–28. 2005. View Article : Google Scholar : PubMed/NCBI

4 

Ayroldi E, Migliorati G, Bruscoli S, Marchetti C, Zollo O, Cannarile L, D’Adamio F and Riccardi C: Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappaB. Blood. 98:743–753. 2001. View Article : Google Scholar : PubMed/NCBI

5 

Ayroldi E, Petrillo MG, Bastianelli A, Marchetti MC, Ronchetti S, Nocentini G, Ricciotti L, Cannarile L and Riccardi C: L-GILZ binds p53 and MDM2 and suppresses tumor growth through p53 activation in human cancer cells. Cell Death Differ. 22:118–130. 2015. View Article : Google Scholar

6 

Ayroldi E, Zollo O, Bastianelli A, Marchetti C, Agostini M, Di Virgilio R and Riccardi C: GILZ mediates the antiproliferative activity of glucocorticoids by negative regulation of Ras signaling. J Clin Invest. 117:1605–1615. 2007. View Article : Google Scholar : PubMed/NCBI

7 

Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, Fleming MD, Schreiber SL and Cantley LC: The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 452:230–233. 2008. View Article : Google Scholar : PubMed/NCBI

8 

Anastasiou D, Yu Y, Israelsen WJ, Jiang JK, Boxer MB, Hong BS, Tempel W, Dimov S, Shen M, Jha A, et al: Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol. 8:839–847. 2012. View Article : Google Scholar : PubMed/NCBI

9 

Hitosugi T, Kang S, Vander Heiden MG, Chung TW, Elf S, Lythgoe K, Dong S, Lonial S, Wang X, Chen GZ, et al: Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Sci Signal. 2:ra732009. View Article : Google Scholar : PubMed/NCBI

10 

Chen Z, Wang Z, Guo W, Zhang Z, Zhao F, Zhao Y, Jia D, Ding J, Wang H, Yao M, et al: TRIM35 interacts with pyruvate kinase isoform M2 to suppress the Warburg effect and tumorigenicity in hepatocellular carcinoma. Oncogene. 34:3946–3956. 2015. View Article : Google Scholar

11 

Chaneton B and Gottlieb E: Rocking cell metabolism: Revised functions of the key glycolytic regulator PKM2 in cancer. Trends Biochem Sci. 37:309–316. 2012. View Article : Google Scholar : PubMed/NCBI

12 

Luo W and Semenza GL: Emerging roles of PKM2 in cell metabolism and cancer progression. Trends Endocrinol Metab. 23:560–566. 2012. View Article : Google Scholar : PubMed/NCBI

13 

Lv L, Xu YP, Zhao D, Li FL, Wang W, Sasaki N, Jiang Y, Zhou X, Li TT, Guan KL, et al: Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol Cell. 52:340–352. 2013. View Article : Google Scholar : PubMed/NCBI

14 

Yang W, Zheng Y, Xia Y, Ji H, Chen X, Guo F, Lyssiotis CA, Aldape K, Cantley LC and Lu Z: ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol. 14:1295–1304. 2012. View Article : Google Scholar : PubMed/NCBI

15 

Yang W, Xia Y, Hawke D, Li X, Liang J, Xing D, Aldape K, Hunter T, Alfred Yung WK and Lu Z: PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. Cell. 150:685–696. 2012. View Article : Google Scholar : PubMed/NCBI

16 

Yang W, Xia Y, Ji H, Zheng Y, Liang J, Huang W, Gao X, Aldape K and Lu Z: Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature. 480:118–122. 2011. View Article : Google Scholar : PubMed/NCBI

17 

Luo W, Hu H, Chang R, Zhong J, Knabel M, O’Meally R, Cole RN, Pandey A and Semenza GL: Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell. 145:732–744. 2011. View Article : Google Scholar : PubMed/NCBI

18 

Lee J, Kim HK, Han YM and Kim J: Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int J Biochem Cell Biol. 40:1043–1054. 2008. View Article : Google Scholar : PubMed/NCBI

19 

Lim J, Hao T, Shaw C, Patel AJ, Szabó G, Rual JF, Fisk CJ, Li N, Smolyar A, Hill DE, et al: A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration. Cell. 125:801–814. 2006. View Article : Google Scholar : PubMed/NCBI

20 

Landschulz WH, Johnson PF and McKnight SL: The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science. 240:1759–1764. 1988. View Article : Google Scholar : PubMed/NCBI

21 

Kouzarides T and Ziff E: Leucine zippers of fos, jun and GCN4 dictate dimerization specificity and thereby control DNA binding. Nature. 340:568–571. 1989. View Article : Google Scholar : PubMed/NCBI

22 

Busch SJ and Sassone-Corsi P: Dimers, leucine zippers and DNA-binding domains. Trends Genet. 6:36–40. 1990. View Article : Google Scholar : PubMed/NCBI

23 

Hashiguchi A, Hitachi K, Inui M, Okabayashi K and Asashima M: TSC-box is essential for the nuclear localization and antiproliferative effect of XTSC-22. Dev Growth Differ. 49:197–204. 2007. View Article : Google Scholar : PubMed/NCBI

24 

Hino S, Kawamata H, Uchida D, Omotehara F, Miwa Y, Begum NM, Yoshida H, Fujimori T and Sato M: Nuclear translocation of TSC-22 (TGF-beta-stimulated clone-22) concomitant with apoptosis: TSC-22 as a putative transcriptional regulator. Biochem Biophys Res Commun. 278:659–664. 2000. View Article : Google Scholar : PubMed/NCBI

25 

Nakamura M, Kitaura J, Enomoto Y, Lu Y, Nishimura K, Isobe M, Ozaki K, Komeno Y, Nakahara F, Oki T, et al: Transforming growth factor-β-stimulated clone-22 is a negative-feedback regulator of Ras/Raf signaling: Implications for tumorigenesis. Cancer Sci. 103:26–33. 2012. View Article : Google Scholar

26 

Gluderer S, Brunner E, Germann M, Jovaisaite V, Li C, Rentsch CA, Hafen E and Stocker H: Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila. J Biol. 9:92010. View Article : Google Scholar : PubMed/NCBI

27 

Kester HA, Blanchetot C, den Hertog J, van der Saag PT and van der Burg B: Transforming growth factor-beta-stimulated clone-22 is a member of a family of leucine zipper proteins that can homo- and heterodimerize and has transcriptional repressor activity. J Biol Chem. 274:27439–27447. 1999. View Article : Google Scholar : PubMed/NCBI

28 

Gao X, Wang H, Yang JJ, Liu X and Liu ZR: Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell. 45:598–609. 2012. View Article : Google Scholar : PubMed/NCBI

29 

Wang HJ, Hsieh YJ, Cheng WC, Lin CP, Lin YS, Yang SF, Chen CC, Izumiya Y, Yu JS, Kung HJ, et al: JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1α-mediated glucose metabolism. Proc Natl Acad Sci USA. 111:279–284. 2014. View Article : Google Scholar

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September 2016
Volume 49 Issue 3

Print ISSN: 1019-6439
Online ISSN:1791-2423

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APA
Liang, F., Li, Q., Li, X., Li, Z., Gong, Z., Deng, H. ... Xiong, W. (2016). TSC22D2 interacts with PKM2 and inhibits cell growth in colorectal cancer. International Journal of Oncology, 49, 1046-1056. https://doi.org/10.3892/ijo.2016.3599
MLA
Liang, F., Li, Q., Li, X., Li, Z., Gong, Z., Deng, H., Xiang, B., Zhou, M., Li, X., Li, G., Zeng, Z., Xiong, W."TSC22D2 interacts with PKM2 and inhibits cell growth in colorectal cancer". International Journal of Oncology 49.3 (2016): 1046-1056.
Chicago
Liang, F., Li, Q., Li, X., Li, Z., Gong, Z., Deng, H., Xiang, B., Zhou, M., Li, X., Li, G., Zeng, Z., Xiong, W."TSC22D2 interacts with PKM2 and inhibits cell growth in colorectal cancer". International Journal of Oncology 49, no. 3 (2016): 1046-1056. https://doi.org/10.3892/ijo.2016.3599