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Potential conflict of interest: Nothing to report.
LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (MAT) gene MAT1A exhibit a chronic reduction in hepatic S-adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe-deficient [SAMe-D]) from MAT1A knockout (MAT1A-KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe-D cells. LKB1 regulates Akt-mediated survival independent of phosphoinositide 3-kinase, adenosine monophosphate protein–activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus-associated ubiquitin-specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p-LKB1(Ser428) in a NASH-HCC animal model (from MAT1A-KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH. Conclusion: The SAMe-D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies. (HEPATOLOGY 2010)
Nonalcoholic fatty liver disease is characterized by triglyceride accumulation in hepatocytes (hepatic steatosis) and steatosis with inflammation (nonalcoholic steatohepatitis [NASH]), which may progress to cirrhosis and hepatocellular carcinoma (HCC).1 It has been demonstrated recently that tumor suppressor genes such as p53, pRb, M6P/IGF2 receptor, and E-cadherin are involved in the development and progression of HCC.2-4 LKB1 (serine/threonine protein kinase 11) is a tumor suppressor whose inactivation by germline or somatic mutations increases the risk of cancer development.5 However, LKB1 can also play an antiapoptotic role in tumor cells with constitutively active Akt6 and can mediate phosphorylation of p53 by decreasing its nuclear import,7 suggesting that LKB1 may be potentially oncogenic. During hepatocyte proliferation and regeneration after partial hepatectomy, hepatocyte growth factor induces LKB1-mediated adenosine monophosphate protein–activated kinase (AMPK) activation, which controls the nucleocytoplasmic shuttling of Hu antigen R (HuR), an RNA-binding protein that leads to an increased half-life of target messenger RNAs (mRNAs) involved in cell cycle progression.8, 9
S-adenosylmethionine (SAMe), the main biological methyl donor and a precursor of glutathione synthesis,10, 11 blocks hepatocyte proliferation and liver regeneration by inhibiting hepatocyte growth factor–induced LKB1/AMPK/HuR activation and cyclin D1 and A2 expression.8, 9 Methionine adenosyltransferase (MAT), which is encoded by the genes MAT1A(expressed in the adult liver) and MAT2A(expressed in extrahepatic tissues and during liver proliferation), catalyzes SAMe synthesis.10, 11MAT1A knockout (MAT1A-KO) mice show a chronic deficiency in hepatic SAMe levels and spontaneously develop NASH and HCC; this resembles the human pathology, in which HCC is characterized by low levels of SAMe and reduced MAT1A expression.12, 13 In addition, hepatic LKB1 and AMPK are activated, cytoplasmic localization of HuR is increased, and levels of several mRNAs involved in cell proliferation are elevated.6, 8
In the present study, we isolated a cell line (SAMe-deficient [SAMe-D]) from MAT1A-KO mouse HCC as a model of a NASH-derived tumor cell. The results obtained in these cells and in human HCC derived from alcoholic steatohepatitis (ASH) and NASH suggest that p53 inactivation by cytoplasmic sequestration and activation of LKB1 can contribute to HCC development.
Procedures performed in MAT1A-KO and wild-type (WT) mice were in compliance with institutional guidelines for laboratory animal use. Fresh tumor specimens with primary HCC from 15-month-old MAT1A-KO mice were enzymatically dissociated,14 and several cell clones of SAMe-D cells were maintained in 10% fetal bovine serum plus Dulbecco's modified Eagle's medium.
Isolation of Hepatocytes.
Hepatocytes were isolated from 3-month-old male WT mice by way of collagenase perfusion (Gibco-BRL).8, 15 Hepatocytes were serum-deprived for 16 hours before ultraviolet C (UVC) treatment at 20 J with a CL-1000UV Crosslinker (254 nm).
RNA Isolation and Real-Time Polymerase Chain Reaction.
Total RNA was isolated using an RNeasy Mini Kit (Qiagen); 1.5 μg of total RNA was retrotranscribed into complementary DNA using SuperScriptIII retrotranscriptase (Invitrogen). Polymerase chain reaction was performed using a BioRad iCycler Thermalcycler.
Protein Isolation and Western Blot Analysis.
Extraction of total protein from cultured cells has been described.9 Cytosolic and membrane lysates from cultured cells were prepared with the subcellular proteome extraction kit (Calbiochem). Thirty micrograms of protein was electrophoresed on sodium dodecyl sulfate–polyacrylamide gels and transferred onto membranes. Membranes were incubated as described in Supporting Table 1.
Surgically resected specimens of 12 patients with liver cirrhosis and HCC were examined. Patients gave informed consent for all clinical investigations, which were performed in accordance with the Declaration of Helsinki. The data and type of biospecimen used were provided by the Basque Biobank for Research with appropriate ethics approval. The cause of liver cirrhosis was considered to be ASH in 10 patients and NASH in 2 patients.
Immunohistochemistry of paraffin sections of formalin-fixed liver samples were performed as described before.9 Images were taken with a 100× objective from an epifluorescence microscope AXIO Imager.D1 (Zeiss).
Cells fixed with ethanol or methanol were blocked for 30 minutes with phosphate-buffered saline containing 0.1% bovine serum albumin, 10% horse serum, and 0.1% Triton and incubated overnight at 4°C with primary antibodies in blocking solution without Triton (Supporting Table 2). Cells were incubated with fluorescein isothiocyanate conjugated to rabbit or mouse immunoglobulin G (IgG) and Hoescht nuclear dye. Cells were examined under a Leica TCS-SP confocal laser microscope using a 60× objective.
SAMe-D cells were transfected with 100 nM small interfering RNA (siRNA) constructs (Qiagen) (Supporting Table 3) using Lipofectamine 2000 (Invitrogen) twice during a 48-hour period (once every 24 hours). Silencing mix was left overnight, and afterward the medium was replaced with fresh 10% fetal bovine plus serum Dulbecco's modified Eagle's medium.
Five hundred micrograms of total cellular protein was immunoprecipitated overnight at 4°C with 10 μg of IgG1 (BD Pharmingen), herpesvirus-associated ubiquitin-specific protease (HAUSP) or Akt (Cell Signaling) antibodies, and protein A Sepharose (Sigma).
RNA immunoprecipitation was performed as described.14 mRNA bound to HuR was measured by way of real-time polymerase chain reaction and normalized with glyceraldehyde 3-phosphate dehydrogenase.
Synthesis of biotinylated transcripts and analysis of HuR bound to biotinylated RNA were performed as described.9, 16
Experiments were performed in triplicate; data are expressed as the mean ± SEM. Statistical significance was estimated using a Student t test.
Characterization of SAMe-D Cells.
In SAMe-D cells, we observed several kinases [p-Akt(Ser473), p-AMPK(Thr172), and p-LKB1(Ser428)], proteins involved in p53 signaling [Mdm2, p-Mdm2(Ser166), Bax, PUMA, and HAUSP], and apoptotic (cleaved caspase-3) and antiapoptotic markers (Bcl-2, HuR) highly expressed compared with WT hepatocytes, which correlated with positive levels of proliferative markers such as cyclin D1 and proliferating cell nuclear antigen (Supporting Fig. 1). Similar results were observed in another SAMe-D cell clone (clone 2) (Supporting Fig. 2A).
In normal cells, expression of p53 is maintained at low levels through control of the ubiquitin proteasome system.17 In SAMe-D cells, however, the p53 gene and protein were overexpressed compared with WT hepatocytes (Supporting Table 4 and Supporting Fig. 1A). Fluorescence in situ analysis in SAMe-D cells revealed p53 genomic amplification with four copies of chromosome 11, each harboring one p53 signal (Supporting Fig. 3), which was not affected by any mutation after sequencing analysis (Supporting Table 5).
Cellular localization can regulate the apoptotic function of p53, and in several human tumors p53 accumulates in the cytoplasm.18-20 p53 was predominantly cytoplasmic in SAMe-D cells and clone 2 (Supporting Figs. 2C, 4A); p53 silencing confirmed the specificity of the staining (Supporting Fig. 4B). Treatment with the nuclear export inhibitor leptomycin B (20 nM) induced the nuclear accumulation of HuR21 but not of p53 (Supporting Fig. 4C), indicating that a hyperactive nuclear export is not responsible for p53 cytosolic localization.
Delay in the Apoptotic Response in SAMe-D Cells.
We then investigated the p53-dependent apoptotic response to short-wavelength UVC irradiation in SAMe-D cells. In SAMe-D cells, cleaved caspase-3 and cytosolic cytochrome c appeared at 12 and 24 hours, respectively, after UVC irradiation; in WT hepatocytes, this response was observed at 2 hours and 1 hour, respectively (Fig. 1A), suggesting that the apoptotic response in SAMe-D cells upon UVC treatment was substantially delayed.
In SAMe-D cells, UVC irradiation increased p53 levels (Fig. 1B), mainly in the cytoplasm (Fig. 1C). A low-level nuclear staining was observed after 6 hours of UVC stimulation, whereas in hepatocytes, p53 was detected exclusively as nuclear speckles after 30 minutes of UVC treatment (Fig. 1C).
Mdm2 is a ubiquitin-protein ligase that regulates the stability of various essential cellular factors, including p21 and p53, as well as its own degradation.22 UVC irradiation induced a sustained decrease of p21 and Mdm2 but a large increase in p53 content despite the active p-Mdm2(Ser166) (Fig. 1B,D,E), suggesting that Mdm2 might not be able to regulate p53 degradation in SAMe-D cells. In addition, an increase of Bax and a decrease of Bcl-xL were observed at the time when apoptosis occurs.
LKB1 and Akt in the Apoptotic Response of SAMe-D Cells.
Akt is a serine/threonine kinase that promotes cell survival and anabolic processes.23, 24 SAMe-D cells expressed high levels of p-Akt(Ser473) that increased after UVC irradiation, peaked at 18 hours, and returned to basal levels after 24 hours (Fig. 2A). In hepatocytes, however, p-Akt returned to basal levels 4 hours after UVC irradiation. Levels of phosphatase and tensin homolog (PTEN) phosphatase, the major negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway,25 were normal in SAMe-D cells (Fig. 2A).
In WT hepatocytes, wortmannin (PI3K inhibitor) and LY294002 (dual mammalian target of rapamycin [mTOR] and PI3K inhibitor) prevented UVC-mediated activation of Akt (phosphorylation at Ser473 and Thr308). Unexpectedly, only a slight attenuation of Akt activation was detected in SAMe-D cells treated with LY294002 or wortmannin (Fig. 2B), suggesting that Akt activation after UVC treatment in SAMe-D cells was PI3K-independent.
mTOR is a PI3K-related protein that regulates Akt phosphorylation by interacting with proteins such as Raptor (forming mTOR complex 1 [mTORC1]) and Rictor (forming mTOR complex 2 [mTORC2]).26 The basal and UVC-stimulated levels of p-mTOR2(Ser2481) were inhibited by LY294002, suggesting that in SAMe-D cells, p-Akt(Ser473) was independent of mTORC2.27 Furthermore, as observed by others,28 wortmannin did not inhibit UVC-induced p-mTOR2(Ser2481) or p-mTOR1(Ser2448) and its downstream effector S6K(Thr389). In contrast, LY294002 inhibited p-mTOR1(Ser2448) and p-S6K(Thr389) (Fig. 2B and Supporting Fig. 5). These data are in agreement with previous studies showing that S6K phosphorylation in response to UVC is mediated through mTOR1 and PI3K but is Akt-independent.29 Rictor silencing did not change p-Akt(Ser473) or p-Akt(Thr308) either at basal levels or after UVC stimulation, and had no effect on wortmannin sensitivity (Fig. 2C and Supporting Fig. 6). In addition, Rictor knockdown down-regulated p-mTOR2 without affecting p-mTOR1 and p-S6K, confirming the high substrate specificity of the two TOR complexes.30
Because SAMe-D cells expressed high levels of p-LKB1(Ser428), we examined its response after UVC treatment in this cell line. UVC-induced LKB1 phosphorylation was independent of PI3K activity (Fig. 2B). Interestingly, LKB1 knockdown reduced p-Akt(Ser473) and p-Akt(Thr308) after UVC treatment and, most importantly, rendered the cells responsive to wortmannin (Fig. 2C and Supporting Fig. 6). In addition, LKB1 silencing did not induce notable changes in p-mTOR2(Ser2481), p-S6K(Thr389) or p-PTEN (Ser370).23, 25, 28 Double silencing of LKB1 and Rictor reduced p-Akt(Ser473 and Thr308) and sensitized the cells to wortmannin, although this effect was not additive compared with only LKB1 silencing. These results are in agreement with the observation that Rictor silencing alone did not affect LKB1 phosphorylation. Importantly, we also observed that the decrease in p-Akt induced by LKB1 knockdown led to an increase in poly(ADP-ribose) polymerase (PARP) cleavage even without apoptotic stimulus (Fig. 2C and Supporting Fig. 6).
Even though AMPK has recently been described as a kinase of Akt,31 UVC did not induce AMPK activation in hepatocytes or in SAMe-D cells (Supporting Fig. 7A). Furthermore, AMPK silencing did not affect Akt activation or render SAMe-D cells responsive to wortmannin, suggesting that this kinase is not involved in this signaling pathway (Supporting Fig. 7B).
Together, these results indicate that the phosphorylation of Akt at Ser473 and Thr308 in SAMe-D cells is not fully dependent of PI3K, mTORC2, or even AMPK, and suggest a parallel pathway involving LKB1 as part of the prosurvival mechanism.
Finally, we studied the extrinsic apoptotic response in SAMe-D cells through stimulation with the Fas agonist Jo2 antibody.32 Although a single dose of Jo2 (2 μg/mL) was enough for the detection of cleaved PARP in primary hepatocytes at 12 hours, SAMe-D cells did not respond to the treatment even at 48 hours (Supporting Fig. 8A). A second dose of Jo2 treatment for an additional 24 hours was necessary to induce cleaved PARP in SAMe-D cells at 48 hours, together with a decrease in p-LKB1(Ser428) and p-Akt(Ser473) (Supporting Fig. 8B).
LKB1 and the Apoptotic Response in SAMe-D Cells.
We observed an increase in nuclear p-LKB1 (Ser428) during the apoptotic response in hepatocytes after UVC treatment, whereas untreated SAMe-D cells showed a mostly basal-cytoplasmic p-LKB1 that moved to the nucleus after UVC stimulus (Fig. 3A, upper panel) and decreased 12 hours later (Fig. 3A, lower panel). This decrease coincided with the apoptotic response observed in these cells.
LKB1 silencing reduced p-Akt(Ser473) in untreated SAMe-D cells, accompanied by a decrease in p-Mdm2(Ser166) and in total Mdm2 levels (Fig. 3B,C). Akt controls the Mdm2/p53 signaling pathway by decreasing Mdm2 proteasomal degradation.33 Consistent with the specific regulation of LKB1 on Mdm2 content, increased levels of nuclear p53 were detected after LKB1 ablation (Fig. 3C). Previous reports have shown that LKB1-mediated phosphorylation of p53(Ser389) regulates its transcriptional activity,7 and that p53 posttranslational modifications play an important role in the stabilization and activation of p53. We observed p-p53(Ser389) in the cytoplasm of unstimulated SAMe-D cells. Hepatocytes showed a nuclear expression 2 hours after UVC treatment (Fig. 3A), whereas in SAMe-D cells, the nuclear staining was observed at 12 hours. After LKB1 knockdown, p-p53(Ser389) was mainly localized in the nuclear compartment in SAMe-D cells (Fig. 3C).
Finally, a reduction in total and active Mdm2 was observed after Akt knockdown in SAMe-D cells, and this effect was accompanied by a slight decrease in p-LKB1 (Fig. 3D). Immunoprecipitation analysis revealed a complex between p-LKB1 and Akt (Fig. 3E) at basal levels in SAMe-D cells, suggesting cross-talk between these two kinases.
HAUSP, a nuclear ubiquitin-specific protease, targets p53 and Mdm2 as substrates and, in concert with Mdm2, plays a dynamic role in p53 functionality.34 SAMe-D cells expressed higher levels of HAUSP than hepatocytes both in the cytoplasm and nucleus (Supporting Fig. 9). Hepatocytes showed predominantly nuclear HAUSP accumulation after UVC treatment (Supporting Fig. 9). A defect in interaction with HAUSP has been shown to cause cytoplasmic accumulation of p53.34 We detected HAUSP-p53 interaction in SAMe-D cells (Fig. 4A), suggesting that this cytosolic HAUSP could be responsible for p53 cytosolic localization in these cells. In fact, we found that HAUSP silencing in SAMe-D cells induced a decrease in levels of Mdm2 (Fig. 4B), the major substrate stabilized by HAUSP,35 and an increase in nuclear localization of p53 after UVC treatment (Fig. 4C, left panel).
LKB1 knockdown in SAMe-D cells also induced a slight increase in the nuclear levels of HAUSP after UVC treatment (Fig. 4C, right panel). This correlated with a reduction in Mdm2 levels, nuclear accumulation of p53, and activation of apoptosis (Fig. 3C). Our findings suggest that localization of HAUSP is dynamically regulated, and it has been shown that phosphorylation in Ser18 and Ser963 could be responsible for this.35
Identification of HAUSP as a Novel Target of HuR.
HuR binds to and regulates many mRNAs that encode stress response, proliferative proteins, and antiapoptotic and apoptotic proteins.21, 36, 37 High HuR levels characterize SAMe-D cells (Supporting Fig. 1), correlating with its prosurvival function during cell division,37 and the promotion of a malignant phenotype.38 Computer analysis of HAUSP mRNA revealed a large 3′ untranslated region (UTR) of 2,000 bp (Fig. 4D, upper panel), suggesting that HAUSP could be a target of HuR. We performed a biotin pull-down with the complementary DNAs corresponding to either the 5′-UTR, the coding region, or the 3′-UTR of HAUSP. We found that HuR was predominantly bound to the 3′-UTR transcript in the specific regions indicated (Fig. 4D, lower panel). Immunoprecipitation assays of HuR-bound mRNAs from hepatocytes and SAMe-D cell lysates revealed an HuR–HAUSP mRNA interaction in both cell types, although the amount of HAUSP mRNA and HuR–HAUSP mRNA complex was clearly elevated in SAMe-D cells compared with hepatocytes (Fig. 4E). Finally, HuR knockdown induced a clear decrease in HAUSP levels, emphasizing that HAUSP is a target of HuR (Fig. 4F).
HuR Activity Is Regulated by LKB1 in SAMe-D Cells.
HuR is predominantly nuclear, and the translocation to the cytoplasm has been linked to its mRNA-stabilizing function.21 We observed a delay in HuR translocation to the cytoplasm in SAMe-D cells compared with hepatocytes after UVC treatment (Fig. 5A). LKB1 knockdown promoted faster cytoplasmic localization of HuR after UVC treatment (Fig. 5B). In summary, our results suggest a novel signaling network between LKB1, HuR, HAUSP, and p53. We identify LKB1 as a key regulator of HuR and HAUSP localization and, consequently, of their functionality, whereas HuR regulates HAUSP mRNA stability.
p53 and p-LKB1 in Livers from MAT1A-KO Mice and Human HCC.
Liver tumors from MAT1A-KO mice highly expressed cytoplasmic p53 and p-LKB1(Ser428) compared with WT mice (Fig. 6A). In HCC samples surgically resected from patients with ASH and NASH, we observed cytoplasmic staining of p53 and p-LKB1(Ser428) (Fig. 6B), suggesting that this localization and activation may be characteristic of HCC in patients with steatohepatitis.
Although LKB1 has been traditionally considered a tumor suppressor,6 the results of this study suggest that LKB1 might play a role in cell survival in liver tumors that have originated from metabolic disorders, such as NASH.
HCC is characterized by low levels of SAMe and reduced expression of MAT1A. Although MAT1A-KO mice represent a chronic model of SAMe deficiency, they develop steatosis, NASH, and HCC in a spontaneous way.8 Liver LKB1 and AMPK are hyperphosphorylated in these animals during the progression of the disease.8 LKB1 localization is predominantly nuclear, and its activation takes place mainly in the cytoplasm.39 In MAT1A-KO mice with HCC, p-LKB1(Ser428) was found to be mostly cytoplasmic. SAMe-D is a cell line isolated from MAT1A-KO mice with HCC as a model of a NASH-derived tumor cell. In this model, the cytoplasmic hyperphosphorylation of LKB1 (Ser 428) prevents UVC-induced apoptosis partially through the Akt survival pathway. In SAMe-D cells, the PI3K inhibitors LY294002 and wortmannin did not affect Akt phosphorylation, and LKB1 depletion was necessary to induce Akt inhibition after wortmannin treatment. We observed an interaction between LKB1 and Akt proteins. In addition, UVC-induced Akt phosphorylation was independent of mTORC2, AMPK, or PTEN activity. Furthermore, LKB1 knockdown induced an increase in PARP cleavage, even without any apoptotic stimuli. These results provide the first evidence of cross-talk between LKB1 and Akt in response to an apoptotic signal, leading us to consider this pathway as a compensatory and salvage mechanism in SAMe-D global response.
It is well known that LKB1 plays a dual role in the regulation of cell death and proliferation through functions linked to the tumor suppressor p53. LKB1 binds to and phosphorylates p53 at Ser389, reducing the efficiency in its nuclear import.39 In SAMe-D cells, total p53 and its phosphorylated form (Ser389) were mostly present in the cytoplasm, although a nuclear accumulation of p-p53 was observed 12 hours after UVC treatment. LKB1 knockdown decreased the amount of p-p53 in SAMe-D cells and increased p53 nuclear accumulation, confirming the existence of cross-talk between these two proteins.
p53 cytoplasmic staining was observed in the HCC of MAT1A-KO mice and in ASH- and NASH-derived human HCC. A defect in HAUSP-p53 interaction has been shown to be a cause of p53 accumulation.40 However, in SAMe-D cells, the levels of HAUSP were higher, and there was a functional interaction between p53 and HAUSP.
LKB1 could play a critical role in the control of the cellular localization of HAUSP in SAMe-D cells. It has been reported that phosphorylation of HAUSP is sufficient to achieve changes in the localization of the enzyme.35 In SAMe-D cells, the partial reduction of LKB1 levels induces nuclear accumulation of the HAUSP compartment after UVC treatment. Thus, LKB1 could be regulating HAUSP localization in SAMe-D cells directly by phosphorylation, or indirectly by regulating interactions with its partners, Mdm2 and p53.34 Consistent with the latter, LKB1 knockdown reduced Mdm2 levels. This could cause a modification in Mdm2 partners and, in this case, alter the localization of HAUSP. In addition, we investigated the influence of HAUSP on the apoptotic response in SAMe-D cells. HAUSP silencing destabilized Mdm2, which is constitutively self-ubiquitinated and degraded in vivo, and led to nuclear accumulation of p53 in SAMe-D cells.
Following the analysis of HAUSP regulation, we identified a translational mechanism involved in this process. For the first time, we have demonstrated that HuR, which is highly expressed in SAMe-D, can specifically bind the 3′-UTR of HAUSP, stabilizing the mRNA and increasing HAUSP levels.
Finally, our findings are summarized in a representative model (Fig. 7). In normal cells, p53 is maintained at low levels, and Mdm2 is responsible for its ubiquitination and degradation. The levels of Mdm2 are limited by its own partner p53 through the transactivation of its gene. In addition, Mdm2 levels are controlled by posttranslational modifications and through the deubiquitinase HAUSP that modulates its stability. In SAMe-D cells, low SAMe levels are due to loss of MAT1A expression, as occurs in different types of HCC. In SAMe-D cells, cytoplasmic LKB1 phosphorylation is maintained at high levels and acts in different ways: (1) phosphorylating Akt that up-regulates p-Mdm2(Ser166), inhibiting its binding to p53; (2) phosphorylating p53(Ser389), making the p53 protein more stable; (3) regulating the cytoplasmic localization of HuR and therefore stabilizing the mRNA of HAUSP; and (4) regulating the cytoplasmic accumulation of HAUSP, allowing HAUSP–p53 interaction and leading to a more stable p53 in the cytoplasm. In summary, LKB1 seems to control the survival pathway through Akt activation and the apoptotic response through p53 and HuR.
Taken together, the SAMe-D cell line is a new model of NASH-derived HCC with a chronic deficiency in MAT1A, in which LKB1 plays a crucial role as principal conductor of a new regulatory mechanism. Our findings might represent a useful tool to uncover new therapeutic strategies for HCC.
We thank Juan Burgos for selection of the human samples and Begoña Rodríguez and Laura Gomez Santos for technical assistance.