Double mutant P53 (N340Q/L344R) promotes hepatocarcinogenesis through upregulation of Pim1 mediated by PKM2 and LncRNA CUDR

P53 is frequently mutated in human tumors as a novel gain-of-function to promote tumor development. Although dimeric (M340Q/L344R) influences on tetramerisation on site-specific post-translational modifications of p53, it is not clear how dimeric (M340Q/L344R) plays a role during hepatocarcinogenesis. Herein, we reveal that P53 (N340Q/L344R) promotes hepatocarcinogenesis through upregulation of PKM2. Mechanistically, P53 (N340Q/L344R) forms complex with CUDR and the complex binds to the promoter regions of PKM2 which enhances the expression, phosphorylation of PKM2 and its polymer formation. Thereby, the polymer PKM2 (tetramer) binds to the eleventh threonine on histone H3 that increases the phosphorylation of the eleventh threonine on histone H3 (pH3T11). Furthermore, pH3T11 blocks HDAC3 binding to H3K9Ac that prevents H3K9Ac from deacetylation and stabilizes the H3K9Ac modification. On the other hand, it also decreased tri-methylation of histone H3 on the ninth lysine (H3K9me3) and increases one methylation of histone H3 on the ninth lysine (H3K9me1). Moreover, the combination of H3K9me1 and HP1 α forms more H3K9me3-HP1α complex which binds to the promoter region of Pim1, enhancing the expression of Pim1 that enhances the expression of TERT, oncogenic lncRNA HOTAIR and reduces the TERRA expression. Ultimately, P53 (N340Q/L344R) accerlerates the growth of liver cancer cells Hep3B by activating telomerase and prolonging telomere through the cascade of P53 (N340Q/L344R)-CUDR-PKM2-pH3T11- (H3K9me1-HP1α)-Pim1- (TERT-HOTAIR-TERRA). Understanding the novel functions of P53 (N340Q/L344R) will help in the development of new liver cancer therapeutic approaches that may be useful in a broad range of cancer types.


INTRODUCTION
Hepatocarcinogenesis is associated with many etiological factors, including hepatitis virus, AFB1, gene mutations. P53 acts as a tumor suppressor and is mutant in human cancers [1] There is growing evidence that mutant p53s promote tumor development, metastasis and progression [2,3,4]. For example, overexpression of mutant p53 (R273H) promotes proliferation of ovarian cancer cell [5] In addition, mutant p53 also induced platelet-derived growth factor receptor β (PDGFRβ) in human liver cancer [6] Further, the wild p53 and mutant p53 proteins show opposing functions in tumor cell motility [7] Moreover, mutant p53 can reduce dicer output [8] and blocks Smad3/N-CoR complex loading on the REGγ promoter region [9] However, the mutant p53 (Y220C) can rescue the function of unstable p53 mutants [10] Some studies indicated that activating transcription factor 3 (ATF3) bound to mutant p53 and then inhibited mutant P53 oncogenic action [11] The Myo10 controls mutant p53 functions in cancers invasion [12] and MDM2 isoforms promotes mutant p53 accumulation [13] Research shows activation of chaperone-mediated autophagy reduces the levels of accumulated mutant p53 [14] and mutant p53 proteins target micro-RNA223 expression in several cancer cells [15]. Intriguingly, mutant p53 mediates metabolic changes (e.g. glycolysis) to promote tumor progress [16,17]. Pyruvate kinase M2 Research Paper www.impactjournals.com/oncotarget (PKM2) has been demonstrated to play a key role in metabolic regulation, gene expression, cell proliferation, cell migration, tumor angiogenesis [18,19]. For examples, nuclear PKM2 acts as a coactivator of β-catenin to induce c-Myc expression and promotes tumorigenesis [20] In this study, we indicate that P53 (N340Q/L344R) promotes hepatocarcinogenesis through upregulation of PKM2. PKM2 tetramer binds to the eleventh threonine on histone H3 (H3T11) that increases the phosphorylation of the eleventh lysine on histone H3 (pH3T11). Moreover, the combination of H3K9me1 and HP1α forms more H3K9me3-HP1α complex which loads onto the promoter region of Pim1, enhancing the Pim1 expression which enhances the expression of Telomerase Reverse Transcriptase (TERT), oncogenic lncRNA HOTAIR and reduces the TERRA expression.

DISCUSSION
To this data, mutant P53 (N340Q/L344R) shows a strong oncogenic function mediated by PKM2 ( Figure  8). P53 (N340Q/L344R) promotes hepatocarcinogenesis through upregulation of PKM2. Both P53 (N340Q/L344R) and PKM2 are upregulated in human hepatocellular carcinoma tissues, and present the positive correlation. And the P53 (N340Q/L344R) promotes the liver cancer cell's growth. Mechanistically, P53 (N340Q/L344R) forms complex with CUDR and the complex binds to the promoter regions of PKM2 which enhances the expression, phosphorylation of PKM2 and its polymer formation. Thereby, the polymer PKM2 (tetramer) binds to the eleventh serine on histone H3 that increases the phosphorylation of the eleventh threonine on histone H3 (pH3T11). Furthermore, pH3T11 blocks HDAC3 binding to H3K9Ac that prevents H3K9Ac from deacetylation and stabilizes the H3K9Ac modification. On the other hand, it also decreased tri-methylation of the ninth lysine ninth on histone3 (H3K9me3) and increases one methylation of the ninth lysine ninth on histone H3 (H3K9me1). Moreover, the combination of H3K9me1 and HP1 α forms more H3K9me3-HP1α complex which binds to the promoter region of Pim1, enhancing the expression of Pim1 that enhances the expression of TERT, oncogenic lncRNA HOTAIR and reduces the TERRA expression. Ultimately, P53 (N340Q/L344R) accerlerates the growth of hepatocellular carcinoma cells by activated telomerase and prolonged telomere through the cascade of P53 (N340Q/L344R) -CUDR-PKM2-pH3T11-(H3K9me1-HP1α)-Pim1-(TERT-HOTAIR-TERRA).
It is worth mentioning that double mutant P53 (N340Q/L344R) may play an important role in the occurrence of liver cancer cancer. In this report, we focused mainly on the view how double mutant P53 (N340Q/ L344R) functions during hepatocarcinogenesis. Although dimeric (M340Q/L344R) influences on the post-translational modifications of p53, it is not clear how dimeric (M340Q/ L344R) plays a role during hepatocarcinogenesis [21]. Our results indicates that P53 (N340Q/L344R) has a strong oncogenic charter. To this date, accumulating evidence indicates mutant P53 function in liver cancer. A large number of modifications on p53 (e.g. 3KR mutant) regulate cellular DNA binding ability and tumor development [22,23,24]. Moreover, several p53 mutant proteins escape proteolytic degradation and exert oncogenic gain-of-function properties [25,26] or causes maintenance of genomic integrity [27,28,28,30] Intruigingly, mutant P53 facilitates dedifferentiation of mature hepatocytes into progenitor-like cells [29] Our present findings are consistent with some reports. Herein  It has been confirmed that PKM2 plays a role in tumor anabolic metabolism [30] For example, knockdown of PKM2 suppressed aerobic glycolysis of liver cancer cell [31] In this study, we found that double mutant P53 (N340Q/L344R) enhanced the pyruvate kinase M2 isoform (PKM2) activity. In fact, PKM2 is required for cell survival and proliferation in tumorigenesis [32,33] Moreover, the PKM2 dimers and tetramers are critical for tumorigenesis and epithelial-mesenchymal transition which is controlled by multiple factors, e.g. miR675 and H19 [34,37] It is worth noting that our findings in this study provide novel evidence for an active role of PKM2 based on double mutant P53 (N340Q/L344R) in liver cancer cell growth. This assertion is based on several observations: (1)Mutant P53 (N340Q/L344R) enhances PKM2 expression and its polymer formation.
Strikingly, our results demonstrates that P53 (N340Q/ L344R) forms complex with CUDR and the complex binds to the promoter regions of PKM2 which enhances the expression, phosphorylation of PKM2 and its polymer formation. In fact, CUDR is closely associated with tumorigenesis. For example, CUDR increased the exprseeion of HULC, β-Catenin, TERT and C-myc in human liver cancer stem cell [36,37]. Moreover, CUDR cooperates with SET1A to trigger stem cell malignant transformation [38].
Of significance, our findings show that double mutant P53 (N340Q/L344R) promotes pim1 expression through H3K9me1 and HP1α dependent on PKM2, and enhances HOTAIR expression, telomerase activity, elongates telomere length in liver cancer cells. This assertion is based on several observations (1)double mutant P53 (N340Q/ L344R) promotes the interplay between H3K9me1 and HP1α. (2)Mutant P53 (N340Q/L344R) enhances Pim1 expression triggered by the increased interplay between H3K9me1 and HP1α. (3)double mutant P53 (N340Q/ L344R) enhances HOTAIR expression and stimulates telomerase activity dependent on pim1. Pim-1 is associated with transcriptional activation [39]. Our previous studies showed that H19 enhanced Pim1 expression and function in the liver cancer [34]. Telomeric repeat-containing RNA (TERRA) is important for telomere regulation [40] and TRF2 represses TERRA transcription through its homodimerization domain [41]. Our previous study showed that the overexpression of H19 increases the binding of TERT to TERC and reduces the interplay between TERT with TERRA, thus enhancing the cell telomerase activity and extending the telomere length in liver cancer stem cells [37]. HOTAIR is is reported to reprogram chromatin organization and promote tumor progression and metastasis [42,43,44]. For examples, HOTAIR could promote Mechanistically, P53 (N340Q/L344R) forms complex with CUDR and the complex binds to the promoter regions of PKM2 which enhances the expression, phosphorylation of PKM2 and its polymer formation. Thereby, the polymer PKM2 (tetramer) binds to the eleventh threonine on histone H3 that increases the phosphorylation of the eleventh lysine on histone H3 (pH3T11). Furthermore, pH3T11 blocks HDAC3 binding to H3K9Ac that prevents H3K9Ac from deacetylation and stabilizes the H3K9Ac modification. On the other hand, it also decreased tri-methylation of the ninth lysine ninth on histone3 (H3K9me3) and increases one methylation of the ninth lysine ninth on histone H3 (H3K9me1). Moreover, the combination of H3K9me1 and HP1 α forms more H3K9me3-HP1α complex. This increased complex binds to the promoter region of Pim1, enhancing the expression of Pim1 that increases the expression of TERT, oncogenic lncRNA HOTAIR and reduced expression of TERRA expression. Ultimately, P53 (N340Q/L344R) accerlerates the growth of hepatocellular carcinoma cells by activated telomerase and prolonged telomere through the cascade of P53 (N340Q/L344R)-CUDR-PKM2-pH3T11-(H3K9me1-HP1α)-Pim1-(TERT-HOTAIR-TERRA). www.impactjournals.com/oncotarget migration and invasion of hepatocellular carcinoma (HCC) cells and promotes human liver cancer stem cell malignant growth [45,46]. In addition, HOTAIR preferentially occupies a GA-rich DNA motif to nucleate broad domains of polycomb occupancy [47] and altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness [48].
We should further explore the function of duble mutant P53 (N340Q/L344R) during hepatocarcinogenesis.
For example, what causes strong oncogenic action duble mutant P53 (N340Q/L344R)? How does duble mutant P53 (N340Q/L344R) do? Does CUDR regulate a series of molecular signaling pathway in liver cancer cells? Answering these questions will help understand the mechanism about hepatocarcinogenesis. Duble mutant P53 (N340Q/L344R) accerlerates the growth of hepatocellular carcinoma cells by activated telomerase and prolonged telomere through the cascade of P53 (N340Q/L344R)-CUDR-PKM2-pH3T11−(H3K9me1-HP1α)-Pim1−(TERT− HOTAIR-TERRA). Understanding the functions of mutant p53 will help in the development of new therapeutic approaches that may be useful in a broad range of cancer types [49].

Cell lines and plasmids
Human hepatoma cell lines Hep3B were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). These cell lines were maintained in Dulbecco's modified Eagle medium (Gibco BRL Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (sigma) in a humidified atmosphere of 5% CO 2 incubator at 37°C. pLVX-Tet-On-Advanced vector and pVSVG were purchased from Cloneth. pVSVG-P53 (N340Q/L344R), PGFP-V-RS-PKM2, pGFP-V-RS-Pim1 were prepared by ourselves.

Cell transfection and stable cell lines
Cells were transfected with DNA plasmids using transfast transfection reagent lipofectamine R 2000 (Invitrogen) according to manufacturer's instructions. For screening stable cell lines, forty-eight hours after transfection, cells were plated in the selective medium containing G418 (2000μg/ml, Invitrogen) for the next 4 weeks or so, and the selective media were replaced every 3 days.

Selection of ShRNA cell lines
Cell lines were transfected with pGFP-V-RS-PKM2, pGFP-V-RS-Pim1 using transfection reagent lipofectamine R 2000 (Invitrogen). Forty-eight hours after transfection, the cells were cultured with the selection media containing 2 μg/ml Puromycin (Invitrogen) for PKM2 or Pim1 knockdown. The selection media were replaced every 3 days.

Tet-On advanced inducible expression
pLVX-Tet-On Advanced System was performed according to the manufacturer's instructions. As to the first stable transfection, the Hep3B cells were transfected with pLVX-Tet-On vector (Clonth) using Lipofecamine TM 2000 (Invitrogen), and then established the stable cell line by selecting and screening using 2mg/ml G418 (BD Biosciences Clontech). To ensure optimal induction and low background, multiple clonal cell lines must be screened after each stable transfection using western blotting. As to the Second stable transfection, pVSVG/ P53 (N340Q/L344R was infected into the pLVX-Tet-On Hep3B stable cell line, and transfected cells were grown in presence of 0.05 mg/ml Zeo (BD Biosciences Clontech) for selection. Individual colonies were isolated and screened for P53 (N340Q/L344R expression. The positive clones were cultured in DMEM medium (Gibco BRL Life Technologies) containing the indicated tetracycline (Tc) (sigma) concentrations (0-2μg/ml) to induce the P53 (N340Q/L344R expression.

RT-PCR
Total RNA was purified using Trizol (Invitrogen) according to manufacturer's instructions. cDNA was prepared by using oligonucleotide (dT) [17][18] , random primers, and a SuperScript First-Strand Synthesis System (Invitrogen). PCR analysis was performed under the specical conditions. β-actin was used as an internal control.

Western blotting
The logarithmically growing cells were washed twice with ice-cold phosphate-buffered saline (PBS, Hyclone) and lysed in a RIPA lysis buffer. Cells lysates were centrifuged at 12,000g for 20 minutes at 4°C after sonication on ice, and the supernatant were separated. After being boiled for 5-10 minutes in the presence of 2-mercaptoethanol, samples containing cells proteins were separated on a 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membranes (Invitrogen, Carlsbad, CA,USA). Then blocked in 10% dry milk-TBST (20mM Tris-HCl [PH 7.6], 127mM NaCl, 0.1% Tween 20) for 1 h at 37°C. Following three washes in Tris-HCl pH 7.5 with 0.1% Tween 20, the blots were incubated with 0.2 μg/ml of antibody (appropriate dilution) overnight at 4°C. Following three washes, membranes were then incubated with secondary antibody for 60 min at 37°C or 4°C overnight in TBST. Signals were visualized by ODYSSEY infrared imaging system (LI-COR, Lincoln, Nebraska USA). IRDye 680LT /IRDye 800CW secondary antibodies were purchased from LI-COR scientific company.

Co-immunoprecipitation (IP)
Cells were lysed in 1 ml of the whole-cell extract buffer A (50mM pH7.6 Tris-HCl, 150mMNaCl, 1%NP40, 0.1mMEDTA,1.0mM DTT,0.2mMPMSF, 0.1mM Pepstatine,0.1mM Leupeptine,0.1mM Aproine). Five-hundred-microliter cell lysates was used in immunoprecipitation with antibody. In brief, protein was pre-cleared with 30μl protein G/A-plus agarose beads (Santa Cruz, Biotechnology, Inc.CA) for 1 hour at 4°C and the supernatant was obtained after centrifugation (5,000rpm) at 4°C. Precleared homogenates (supernatant) were incubated with 2 μg of antibody and/ or normal mouse/rabbit IgG by rotation for 4 hours at 4°C, and then the immunoprecipitates were incubated with 30μl protein G/A-plus agarose beads by rotation overnight at 4°C, and then centrifuged at 5000rpm for 5 min at 4°C. The precipitates were washed five times×10min with beads wash solution (50 mM pH7.6 TrisCl,150mMNaCl,0.1%NP-40,1mM EDTA) and then resuspended in 60μl 2×SDS-PAGE sample loading buffer to incubate for 10 min at 100°C. Then Western blot was performed with a another related antibody indicated in Western blotting.

Chromatin immunoprecipitation (CHIP) assay
Cells were cross-linked with 1% (v/v) formaldehyde (Sigma) for 10 min at room temperature and stopped with 125 mm glycine for 5 min. Crossed-linked cells were washed with phosphate-buffered saline, resuspended in lysis buffer, and sonicated for 8-10 min in a SONICS VibraCell to generate DNA fragments with an average size of 500 bp or so. Chromatin extracts were diluted 5-fold with dilution buffer, pre-cleared with Protein-A/G-Sepharose beads, and immunoprecipitated with specific antibody on Protein-A/G-Sepharose beads. After washing, elution and de-cross-linking, the ChIP DNA was detected by either traditional PCR.

Super-EMSA (Gel-shift)
Cells were washed and scraped in ice-cold PBS to prepare nuclei for electrophoretic gel mobility shift assay with the use of the gel shift assay system modified according to the manufacturer's instructions (Promega). In brief, consensus oligonucleotides for damage or repair DNA was biotin-labeled (hot probe). Each binding reaction was carried out with 1 μg biotinylated dsDNA probe and 200 μg purified nuclear protein in 20 μl of binding buffer containing 0.5mg/ml poly (dI:dC) (25 mM HEPES at PH8.0 with 50 mM KCl. 0.1% Triton X100, 2 mM MgCl2, 3 mM DTT, and 5% glycerol). Twenty-five pmol unlabeled cold DNA motifs (a 250-fold excess) were added in the competition assays. Reactions were carried out for 30 min incubation at room temperature, followed by overnight incubation at 4°C. Reaction mixtures were loaded onto 6% TBE polyacrylamide gels and separated in 0.5×TBE at 100v on ice until the dye front migrated twothirds of the way to NC membranes and Western blotting for anti-biotin.

Dual luciferase reporter assay
Cells (1x10 5 /well of a six-well plate) were transiently transfected by use of the Lipofectiamine TM 2000 (Invitrogen). After incubation for 48 h, the cells were harvested with Passive Lysis Buffer (Promega), and luciferase activities of cell extracts were measured with the use of the Dual luciferase assay system (Promega) according to manufacturer's instructions. luciferase activity was measured and normalized for transfection efficiency with Renilla luciferase activity.

Quantitative telomerase detection
The telomerase activity was measured byusingQuantitative Telomerase Detection Kit (MT3010) according to manufacturer's instructions (US Biomax, Inc). In brief, Resuspend the cell pellet in 200 μl of 1× Lysis Buffer /10 5 -10 6 cells. Incubate the suspension on ice for 30 minutes. Spin the sample in a microcentrifuge at 12,000x g for 30 minutes at 4°C. Transfer 160 μl of the supernatant into a fresh tube and determine the protein concentration. Mix the 2×master mix thoroughly and dispense appropriate volumes into PCR thin-wall PCR plates. Add 1 μl of test extract, heat-inactivated extracts or template controls to the individual PCR tubes containing the master mix PCR Initial 10 min 95°C HotActivited Tag DNA Polymerase. Activation Step is activated by this heating step 3 -step cycling:Denaturation 30s 95°C;Annealing 30 s 60°C;Extention 30 s 72°C. Cycle number 40 cycles Cycle. The PCR Quantification screen is displayed during the PCR run and presents data as they are being collected in real time. Collect the threshould cycle or CT value after cycles finished. The threshould cycle is the cycle at which a statistically significant increase in Δ Rn is first detected. Threshold is defined as the average standard deviation of Rn for the early cycles, multiplied by an adjustable factor. A standard curve was generated using the reading of the threshold (CT) of Real-Time PCR.

Telomere length assay
Telomere length assay using Telo TAGGG PCR ELISApuls kit according to manufacturer's instructions (Roche). A standard curve is established by dilution of known quantities of a synthesised 84 mer oligonucleotide containing only TTAGGG repeats.

Nuclear run on assay
Nuclear run-on was performed by supplying biotinprobe to nuclei, and labeled transcripts were bound to streptavidin-coated streptavidin-agarose Resin [50] The cells are chilled, and the membranes are permeabilized or lysed. The nuclei are then incubated for a short time at 37°C in the presence of nucleoside triphosphates (NTPs) and biotin labeled probe. The number of nascent transcripts on the gene at the time of chilling is thought to be proportional to the frequency of transcription initiation.
To determine the relative number of nascent transcripts in each sample, the biotin labeled RNA is purified and hybridized to a membrane containing immobilized DNA from the gene of interest. The amount of biotin activity that hybridizes to the membrane is approximately proportional to the number of nascent transcripts.

Cells proliferation CCK8 Assay
Cells were synchronized in G0 phase by serum deprivation and then released from growth arrest by reexposure to serum, and then cells were grown in complete medium for assay. The cell proliferation reagent CCK8 is purchased from Roch and the operation according to the manufacturer instruction.
Soft agar colony formation capacity assay 2 x 10 2 cells were plated on a 6 well plate containing 0.5% (lower) and 0.35% (upper) double layer soft-agar. The 6 well plates were incubated at 37°C in humidified incubator for 14 days. The cells were fed 1-2 times per week with cell culture media (DMEM). Soft-agar colonies on the 6 well plates were stained with 0.5 ml of 0.05% Crystal Violet for more than 1 hour and the colonies were counted.

BrdU staining
70-80% confluent cells were cultured for 24 hour before treatment with BrdU (Roche) for 4 hours. Immunofluorescent staining with an anti-BrdU antibody was performed according to the manufacturer's instructions (Becton Dickinson). BrdU positive cells from ten random chosen fields of at least three independent samples were counted.

Xenograft transplantation in vivo
TheFour-weeks athymic Balb/C mouse was injected the liver cancer cells at the armpit area subcutaneously. The mice were observed over 4 weeks, and then sacrificed to recover the tumors. The wet weight of each tumor was determined for each mouse. A portion of each tumor was fixed in 4% paraformaldehyde and embedded in paraffin for histological hematoxylin-eosin (HE) staining. The use of mice for this work was reviewed and approved by the institutional animal care and use committee in accordance with China national institutes of health guidelines.