MEK1 Signaling Promotes Self-renewal And Tumorigenicity Of Liver Cancer Stem Cells Via Maintaining SIRT1 Protein Stabilization

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in China.Although specific surgery has been established as the first-line treatment for HCC accompanied with other therapy, HCC patients still suffer from the high morbidity of postoperative recurrence and therapy resistance which both lead to an indefinite survival time.This high mortality has been commonly attributed to the presence of residual cancer stem cells (CSCs). For these cells have tumor initiating properties and self-renewal function like embryonic stem cells, which also display a role of differentiation that helps to resist drug and radiation therapy. Previous studies show that CSCs play a key role in tumorigenesis, cancer metastasis, therapy resistance and tumor relapse after surgery. More and more researches of CSCs reveal their specific function mechanism, which not only help to make a better understanding of cancer, but also provide new hope for overcoming cancer.Our previous study demonstrated that transcription factor Nanog acted as a CSCs marker which regulated self-renewal through IGF-1 signaling in HCC, no matter in vitro or vivo conditions. Those HCC stem cells are mainly responsible for HCC patients’ bad prognosis,for their capable of sustaining liver cancer self-renewal, formation and differentiation.Therefore, we can not elaborate the underlying mechanisms of CSCs function in HCC growth and relapse.The sirtuins (silent mating type information regulators) is a highly conserved family of NAD-dependent enzymes. It contains seven family members (SIRT1-7), which control various cellular processes including cell cycle, cellular metabolism, cell proliferation,differentiation, genome stability and tumor formation. Simultaneously, SIRT1 (Silent information regulator 1) is regarded as a key molecular in HCC maintenance and development. Although we find that expression of microRNA (miRNA)-34a, miRNA-29c and c-MYC can regulate oncogenic SIRT1 in HCC, the particular mechanism about regulation of HCC stem cells through SIRT1 has not been clear.MEK1 and MEK2 belong to the family of MAPKKs. They are dual specificity enzymes that can phosphorylate threonine and tyrosine residues, accompanying with the activation of their MAP kinase downstream substrates. Dysregulation of MEK1 has been implicated in many diseases, including cancer. MEK1 is up-regulated in a number of tumors’ genesis. Meanwhile, MEK1 signaling is regarded as a key molecular in HCC maintenance and development. However, nobody has figured out the particular mechanisms that how MEK1 signaling regulates liver CSCs self-renewal.In fact, our studies show a novel mechanism that MEK1 inactivation inhibits HCC tumorigenesis in vitro and vivo by promoting SIRT1 ubiquitination which results in SIRT1 protein degradation. To reach those purposes, we separated this problem into four sections.Firstly, we proved that MEK1 played an important role in liver CSCs proliferation and self-renewal. Then, we found MEK1 was significantly correlated with SIRT1 in HCC patients and MEK1 inhibition/knockdown significantly down-regulated SIRT1. Next, we elucidated that MEK1 regulated SIRT1 by maintaining protein degradation, which also certified that MEK1 signaling regulated liver CSCs self-renewal via controlling SIRT1 protein level. Finally, we surprisingly clarified that MEK1/SIRT1 expression level was associated with HCC patients’prognosis.The main results and conclusions of my study are presented as follows1. Inhibition of MEK1 activity can significantly decrease liver cancer stem cells proliferation(1)Huh7-NanogPos and PLC/PRF/5-NanogPos cells under different U0126 concentrations treatment were seeded (1×103) and cultured for another 6 days before analyzed with CCK-8.We found that U0126 significantly inhibited liver CSCs proliferationin dose-dependent manner. Cell survival analysis showed that these doses didn’t significantly kill liver CSCs with poison.(2)Huh7-NanogPos and PLC/PRF/5-NanogPos cells were cultured with U0126 or DMSO for 48 hours. Immunofluorescence (IF) analysis showed that Ki-67 was dramatically reduced in liver CSCs after treatment with U0126 compared with control group.(3)Cell cycle profiles of U0126 treated or DMSO treated (negative control) Huh7- and PLC/PRF/5-NanogPos cells followed by treatment with sodium butyrate. U0126 treatment caused a significant reduction of S and G2/M phases and increase of G0/G1 phase in liver CSCs.2. MEK1 inhibitor U0126 can significantly decrease liver CSCs self-renewal(1) MEK1 activity inhibition dramatically decreased liver CSCs sphere formation efficiency and clone formation efficiency in two cell lines. Contrary to liver CSCs, low concentration of U0126 treatment did not reduce clone and sphere formation efficiency on liver non-CSCs.(2)Western blot analysis showed that inhibition of MEK1 activity in liver CSCs markedly restrained expression of sternness marker such as SOX2, OCT4 and Nanog.(3) U0126 inhibition liver CSCs can significantly prevent tumor growth, when we subcutaneous injected 1×102, 1×103, 1×104 cells into NOD-SCID mice.3. MEK1 knock-down can significantly decrease liver CSCs self-renewal and proliferation characteristics(1)Western blot analysis showed that MEK1 and phospho-ERK 1/2 level was significant decreased when knockdown of MEK1 expression in liver CSCs, while ERK1/2 expression remained the same.(2) MEK1 knockdown with two individual lentiviruses significantly inhibited liver CSCs proliferation.(3) MEK1 knockdown significantly inhibited liver CSCs clone and sphere formation efficiency compared to negative control.(4) Western blot results showed that MEK1 knockdown significantly inhibited expression of sternness-related proteins, including Nanog, OCT4, c-Myc and SOX2.4. MEK1 inhibition/knockdown can significantly decrease SIRT1 expression(1)Western blot results indicated that SIRT1 and SIRT7 expression were higher in liver CSCs than non-CSCs, and inhibition of MEK1 activity significantly decreased SIRT1 expression.(2) Treatment of liver CSCs with U0126 decreased SIRT1 protein level at a dose ortime-dependent manner, just as Western blot results.(3) Another MEK1 inhibitor (PD98059) proved that MEK1 activity inhibition suppressed SIRT1 protein expressionin liver CSCs.(4) MEK1 knockdown inhibited SIRT1 protein expression efficiently in liver CSCs.5. MEK1 signaling promotes liver CSCs self-renewal and tumorigenicity relaying on histone deacetylase SIRT1 expression(1) SIRT1 overexpression increased clone and sphere formation efficiency in liver non-CSCs, but these effects were reversed by the MEK1 activity inhibition.(2) MEK1 inhibition down-regulated the efficiency of colony or sphere in CSCs,which was then rescued by reconstituted expression of SIRT1 protein.(3) MEK1 inhibition significantly inhibited liver CSCs tumor formation in NOD-SCID mice compared with CSCs. Meanwhile, overexpression SIRT1 protein partly recovered liver CSCs tumorigenicity in vivo condition.6. MEK1 maintain SIRT1 protein level via inhibiting SIRT1 ubiquitination level to suppress proteasome(1) We co-cultured proteasome inhibition (MG-132), MEK1 inhibition or knockdown liver CSCs with CHX (Cycloheximide) , then Western blotsresults showed that MEK1 inhibition or knockdown sped up SIRT1 protein degradation, compared to negative control.On the other hand, MG-132 slowed down process of SIRT1 degradation.(2) MEK1 knockdown or inhibition in liver CSCs significantly promoted SIRT1 protein degradation via activating proteasome activity.(3) Co-IP results proved that MEK1 knockdown or inhibition in liver CSCs up-regulated SIRT1 ubiquitination statement, which led to SIRT1 degradation.7. High expression of MEK1/SIRT1 in human HCC patients was positively associated with poor prognosis(1) p-MEK1 (phosphorylated-MEK1), SIRT1 and Nanog protein expression were detected by Immunohistochemistry (IHC) analysis in 148 HCC patients. It was clear that high expression of p-MEK1/SIRT1 was 48 patients, while low expression of p-MEK1/SIRT1 was 39 patients. Person chi-square test showed P value was 0.035, meant MEK1 correlated to SIRT1 in HCC patients. Meanwhile, the correlation between Nanog expression and MEK1/SIRT1 in tumor tissues was significantly observed (Pearson Correction=0.013/0.038).(2) By gathering and analyzing clinicopathological parameters in the 148 HCC patients, we found that there were no statistical correlation between MEK1/SIRT1 expression and some clinicopathological parameters, such as patient age, gender, AFP level in serum, tumor interstitial hyperplasia, necrosis and recurrence. However, we found the remarkable positive correlation between MEK1/SIRT1 expression and tumor size (p=0.012),vascular thrombus (p<0.001), capsular invasion (p=0.048) and clinical tumor stage (p<0.001).(3) Kaplan-Meier’s analysis revealed that MEK1 and SIRT1 expression in HCC was significantly correlated with prognosis and survival time. Moreover, patients who were combination of high MEK1 and SIRT1 expression had the poorer outcome compared with the low expression group.Taken together, our experiment data reveal that1. MEK1 expression is associated with SIRT1 level in HCC patients, and co-expression of MEK1/SIRT1 in high statement for HCC patients always lead to poor outcome and limited lifetime.2. MEK1 activation promote liver cancer stem cells proliferation, self-renewal and tumorigenic properties. MEK1 knockdown or inhibition significantly inhibits those stemness characteristics.3. MEK1 regulated liver cancer stem cells proliferation, self-renewal and tumorigenisis through SIRT1 protein expression.4. MEK1 maintains SIRT1 expression via controlling proteasome degradation.5. MEK1/SIRT1 can server as a marker to diagnosis HCC patients’ prognosis, and MEK1 signaling can be a potential therapy target for HCC patients.All in all, we show that inhibition or depletion of MEK1 can significantly decrease liver CSCs self-renewal and tumor growth both in vitro and vivo conditions. Furthermore,we demonstrate that MEK1 signaling promotes liver CSCs self-renewal and tumorigenicity by maintaining SIRT1 level. Mechanistically, MEK1 signaling keeps SIRT1 protein stabilization through inhibiting SIRT1 ubiquitination, which inhibits proteasomal degradation. Clinical analysis shows that patients co-expression of MEK1 and SIRT1 are associated with poor survival. Our finding indicates that MEK1-SIRT1 can act as a novel diagnostic biomarker and inhibition of MEK1 may be a viable therapeutic option for targeting liver CSCs’ treatment.