The Effects And Mechanisms Of MUC1Promoting Hepatocellular Carcinoma Progression Through Converting TGF-β Signaling From Tumor-suppression To Oncogenesis

Mucin1(MUC1) as an oncogene could promote adenocarcinoma formation andprogression by regulating multiple proliferation-promoting and apoptosis-inhibitingsignaling. However, whether MUC1mediates TGF-β signaling in cancer cells has notpreviously been reported. Our previous study has found that MUC1gene silencing notonly decreased the malignancy, but also decreased transforming growth factor beta(TGF-β) signaling by global gene expression analysis in the human hepatocellularcarcinoma (HCC) cell line SMMC-7721, suggesting a new mechanism that MUC1maypromote cancer progression by mediating TGF-β signaling. TGF-β signaling plays atumor suppressive role in normal epithelia and precancerous tissue, but accelerates theprogression of established cancers. However, the mechanisms underlying this dual role ofTGF-β signaling are still unclear. Recently, studies have shown that intracellular kinasescan differentially phosphorylate the Smad at their C-terminal and linker region to becomecanonical Smad pathway and Non-canonical Smad pathway and then transmittumor-suppressive or oncogenic TGF-β signaling, respectively, by mediating distincttranscriptional responses. Moreover, JNK activation can activate Non-canonical Smadpathway, and then shifting TGF-β signaling from tumor-suppression to oncogenesis.Furthermore, MUC1can inhibit colorectal cell apoptosis by activating JNK, leading tothe hypothesis that MUC1may shift TGF-β signaling from tumor-suppression tooncogenesis by activating JNK to promote HCC progression.In this study, we silenced and over-expressed MUC1gene in HCC cell lines toinvestigate the effect of MUC1on cell proliferation and TGF-β signaling, and then investigating the role of JNK in MUC1-mediated TGF-β signaling by blocking signaltransduction, RNA interference, luciferase reporter assay and immunoprecipitation assay.Further, we verified the relationship among MUC1, TGF-β signaling and JNK in vivoand tumor tissues from HCC patients.Our studies include the following contents:Establishing MUC1gene silencing and overexpressing HCC cell lines1. Identification of MUC1-silenced stable cell clonesThe MUC1gene was silenced in HCC cell line SMMC-7721using RNAi. Twoindependent MUC1-knockdown stable cell clones, named MR1-D4and MR1-D9, and anegative control clone, named NC, were analyzed by immunofluorescence staining andWestern blotting. The results showed that MUC1expression in MR1-D4and MR1-D9cell clones was significantly decreased compared to NC or SMMC-7721cells. Thesilencing efficiency in clones MR1-D4and MR1-D9reached82.34%and80.13%,respectively.2. Establishing MUC1overexpressing HCC cell linesBoth Bel-7402and Hep3B cells, which almost do not express MUC1, wereinfected with a lentivirus construct inserting full-length human MUC1. Then, MUC1expression was identified by flow cytometry and Western blotting. Two MUC1overexpressing cell lines, named7402-MUC1and Hep3B-MUC1, and two negativecontrols, named7402-EV and Hep3B-EV, were established. The results showed thatMUC1expression efficiency in7402-MUC1and Hep3B-MUC1cell lines reached99.62%and99.53%, respectively.The effect of MUC1on HCC cell proliferation1. The effect of MUC1on HCC cell proliferation in vitroTo investigate the influence of MUC1on HCC cell proliferation, we performedWST-1cell viability assay. The result showed that the cell viability of MUC1-knockdownMR1-D4and MR1-D9cells was significantly reduced compared to NC or SMMC-7721cells. In contrast, MUC1overexpression in Bel-7402and Hep3B cells increased the cellviability compared to the control cells. The result demonstrates that MUC1enhances HCC cell proliferation in vitro.2. The effect of MUC1gene silencing on tumorigenic capacity of HCC cells in nudemiceTo evaluate the effect of MUC1on HCC progression in vivo, SMMC-7721, NC,MR1-D4and MR1-D9cells were inoculated subcutaneously into BALB/c nude mice toestablish a subcutaneous transplant tumor model. Twenty-one days after tumorimplantation, the mice were observed by an in vivo imaging system. The result showedthat the MR1-D4and MR1-D9groups grew smaller tumors compared with the NCgroups. This result indicates that MUC1gene silencing significantly inhibits the growth ofSMMC-7721cells in vivo.The effect of MUC1on TGF-β signaling of HCC cellsTo investigate the influence of MUC1on TGF-β signaling of HCC cells, weperformed qRT-PCR assay to detect mRNA levels of Smad-dependent signalingmolecules such as TGF-β1, TGF-β2, TGF-β3, TβRI, TβRII, Smad2, Smad3, Smad4, andSmad7, and Smad-independent signaling molecules such as RhoA and RhoB in MUC1gene silencing and overexpressing HCC cell lines. The result showed that TGF-β1,TGF-β2, Smad2and Smad3mRNA levels were down-regulated in MUC1-knockdownMR1-D4and MR1-D9cells compared to the control cells. In contrast, MUC1overexpression in Bel-7402and Hep3B cells increased TGF-β1, Smad2and Smad3mRNA levels. The result demonstrates that MUC1mediates Smad-dependent TGF-βsignaling in HCC cells.MUC1mediates autocrine TGF-β signaling through activating the JNK/AP1pathway1. MUC1mediates autocrine TGF-β signaling in HCC cellsOur previous study showed that MUC1gene silencing decreased the malignancy ofHCC cells, and Smad3as a central mediator mainly transmits proliferation-associatedTGF-β signaling; to investigate whether MUC1regulates TGF-β signaling, Smad3expression and activation in MUC1gene silencing and overexpressing HCC cells weredetected by Western blotting analysis. The levels of Smad3and phosphorylated Smad3L (Ser-213) were significantly decreased in MUC1-knockdown MR1-D4and MR1-D9cells compared to the control cells. In contrast, MUC1overexpression in Bel-7402andHep3B cells elevated Smad3expression and Smad3L (Ser-213) activation. Wespeculated that MUC1might induce TGF-β secretion resulting in up-regulation of TGF-βsignaling. The TGF-β1protein level and promoter transcription level were measured byELISA and luciferase reporter assays. The results showed that TGF-β1protein andpromoter transcription levels were down-regulated in MUC1-knockdown MR1-D4andMR1-D9cells compared to the control cells. In contrast, MUC1overexpression inBel-7402and Hep3B cells elevated TGF-β1protein and promoter transcription levels.These results demonstrate that MUC1enhances luciferase activity derived by a TGF-βpromoter and mediates TGF-β secretion and downstream signal transduction in HCCcells.2. The mechanism of MUC1inducing autocrine TGF-β signalingSome studies have reported that JNK can mediate TGF-β secretion throughactivating AP1, a transcription factor that principally comprises members of the Jun andFos nuclear oncoprotein families. Therefore, we speculated that MUC1may up-regulateTGF-β transcription via activation of the JNK/AP1pathway. Western blotting analysisshowed that the levels of phosphorylated JNK and c-Jun, a member of AP-1transcriptionfactor families, were significantly inhibited in MUC1-knockdown MR1-D4and MR1-D9cells compared to the control cells. In contrast, MUC1overexpression in Bel-7402andHep3B cells elevated JNK and c-Jun activation. Both the JNK inhibitor (SP600125) andthe AP1inhibitor (Curcumin) inhibited MUC1-induced autocrine TGF-β signaling. Theseresults indicate that MUC1induces autocrine TGF-β signaling through activating theJNK/AP1pathway.3. MUC1directly binds and activates JNK in HCC cellsTo thoroughly investigate the mechanism of MUC1activating JNK, we performedimmunofluorescence confocal microscopy assay. The co-location of MUC1and JNK wasobserved with confocal fluorescence microscopy. Further, co-immunoprecipitation analysis showed that MUC1-CT was detected and directly bound to JNK in7402-MUC1cells. These results indicate that MUC1directly binds and activates JNK in HCC cells.4. The effect of exogenous TGF-β signaling on JNK activationRecent studies have shown that TGF-β can activate JNK by mediatingSmad-independent signaling. To additionally evaluate the effect of MUC1-inducedTGF-β secretion on JNK activation, we treated cells with exogenous TGF-β1and TGF-βreceptor (TβR) inhibitor (SB431542) and performed Western blotting analysis. Theeffects of exogenous TGF-β1and TβR inhibitor on JNK phosphorylation were notobserved in the MUC1-overexpressing cells or control cells. These results further indicatethat JNK activation is dependent on MUC1but not MUC1-induced TGF-β in HCC cells.5. MUC1-induced autocrine TGF-β signaling promotes HCC cell migrationTo further study the effect of MUC1-induced autocrine TGF-β signaling on HCCcells, we treated7402,7402-EV and7402-MUC1cells with exogenous TGF-β and thendetected the expression of p-Smad3(Ser-213) and MMP-9, a migration-related targetgene of TGF-β signaling, and determined cell migration by Transwell assays. The resultsshowed that MUC1overexpression in Bel-7402cells increased the levels of p-Smad3L(Ser-213) and MMP-9and promoted cell migration, which was similar to the effect ofexogenous TGF-β1on7402-EV cells. When the TβR inhibitor was added,MUC1-induced Smad3phosphorylation and MMP-9expression were almost abolished,and cell migration was significantly inhibited. These results demonstrate that MUC1promotes cell migration by mediating autocrine TGF-β signaling in HCC cells.6. The effect of TGF-β1gene silencing on MUC1-induced autocrine TGF-βsignalingTo deeply certificate that MUC1induced autocrine TGF-β signaling in HCC cells,TGF-β1gene was silenced by siRNA in7402-MUC1cells. The result showed that thesilencing efficiency of TGF-β1gene induced by TGF-β1-siRNA1and TGF-β1-siRNA2reached61.58%and82.76%respectively compared with NC siRNA. Moreover, TGF-β1gene silencing markedly inhibited MUC1-induced Smad3signaling and cell migration.These results demonstrate that MUC1mediates autocrine TGF-β signaling in HCC cells. MUC1shifts Smad3signaling from the tumor-suppressive p-Smad3C/p21pathwayto the oncogenic p-Smad3L/c-Myc pathway by activating JNK in HCC cells1. MUC1activates p-Smad3L/c-Myc pathway and inhibits p-Smad3C/p21pathwayStudies have shown that the roles of TGF-β signaling are dependent on Smad3phospho-isoforms in tumor progression; to investigate whether MUC1regulates Smad3signaling, Smad3phospho-isoforms and the corresponding target gene expression weredetected by Western blotting in HCC cells. The results showed that the levels ofphosphorylated Smad3L (Ser-213) and its target gene c-Myc were significantly decreased,but the levels of phosphorylated Smad3C (Ser-423/425) and its target gene p21weresignificantly elevated in the MUC1knocked down MR1-D4and MR1-D9cells comparedto the NC or SMMC-7721cells. In contrast, p-Smad3L (Ser-213) and the c-Mycexpressions were upregulated, but p-Smad3C (Ser-423/425) and p21expressions weredownregulated in MUC1-overexpressing Bel-7402and Hep3B cells compared to thecontrol cells. These results indicate that MUC1shifts Smad3signaling from atumor-suppressive p-Smad3C/p21pathway to an oncogenic p-Smad3L/c-Myc pathway.2. JNK inhibitor inhibits p-Smad3L/c-Myc pathway and activates p-Smad3C/p21pathway in HCC cellsBecause JNK is a key conductor in the switch in Smad3signaling, and our resultshave shown that MUC1can active JNK, to further clarify the mechanism by whichMUC1shifts Smad3signaling, we administered MUC1-overexpressing Bel-7402withthe JNK inhibitor SP600125, and performed WST-1cell viability and Western blottingassays. The results showed that when SP600125was administered, cell viability wasinhibited, and the levels of p-Smad3L (Ser-213) and c-Myc were decreased, whereas thelevels of p-Smad3C (Ser-423/425) and p21were elevated. These results indicate thatMUC1activates JNK and then shifting Smad3signaling from a tumor-suppressivep-Smad3C/p21pathway to an oncogenic p-Smad3L/c-Myc pathway in HCC cells. 3. Exogenous TGF-β signaling has no effect on the MUC1-induced switch in Smad3signalingTo evaluate the effect of MUC1-induced TGF-β secretion on the MUC1-inducedswitch in Smad3signaling, the7402-EV and7402-MUC1cells were treated withexogenous TGF-β1and TβR inhibitor (SB431542). Then, the cell viability was analyzedby WST-1, and Smad3phospho-isoforms were detected by Western blotting. The effectsof exogenous TGF-β1and TβR inhibitor on cell proliferation were not observed in theMUC1-overexpressing cells. Moreover, TGF-β1enhanced the levels of both p-Smad3L(Ser-213) and p-Smad3C (Ser-423/425), which were inhibited by the TβR inhibitor inboth7402-EV and7402-MUC1cells; exogenous TGF-β1and TβR inhibitor had noeffect on the MUC1-induced switch in Smad3signaling. These results further indicatethat MUC1modulates Smad3signaling by directly activating JNK in HCC cells.The correlation assay among MUC1, TGF-β signaling and p-JNK expression inBALB/c nude miceSMMC-7721, NC, MR1-D4and MR1-D9cells were inoculated subcutaneously intonude mice to establish a subcutaneous transplant tumor model. Twenty-one days aftertumor implantation, the mice were killed, and the tumors were dissected.Immunohistochemical staining showed that MUC1, p-JNK, p-Smad3L (Ser-213) andc-Myc expressions were highly positive in tumor tissues from the SMMC-7721or NCgroups but were weakly positive or even negative in tumor tissues from the MR1-D4andMR1-D9groups. These results indicate a correlation between MUC1and thep-JNK/p-Smad3L/c-Myc pathway expression in mice.The correlation assay among MUC1, TGF-β signaling and p-JNK expression intumor tissues from HCC patientsNormal liver tissues from hemangioma patients and tumor tissues from HCCpatients were collected and analyzed preliminarily by immunohistochemical staining.MUC1, TGF-β1and p-JNK/p-Smad3L/c-Myc expression were highly positive in tumortissues but weakly positive in normal liver tissues. However, p-Smad3C/p21expressionswere weakly positive in tumor tissues but highly positive in normal liver tissues. The result indicates a high positive correlation among MUC1, TGF-β1andp-JNK/p-Smad3L/c-Myc expression, and a reverse correlation between MUC1andp-Smad3C/p21expression in HCC patients.ConclusionIn conclusion, our results indicate that MUC1mediates autocrine TGF-β signalingthrough directly activating the JNK/AP1pathway to promote HCC cells migration.Moreover, MUC1shifts Smad3signaling from the tumor-suppressive p-Smad3C/p21pathway to the oncogenic p-Smad3L/c-Myc pathway by activating JNK to enhance HCCcells proliferation, revealing a novel pathway by which MUC1acts as an oncogene toregulate tumor development through TGF-β signaling, indicating that oncogenes not onlyalter intracellular signaling in cancer cells but also possesses the proficiency to mediatethe tumor microenvironment through TGF-β, and suggesting that MUC1is an importanttarget for HCC therapy.