The Position And Role Of Nanog Positive Cells In The Differentiation Hierarchy Of Liver Cancer Stem Cells

Liver cancer is the fifth leading cause of cancer-related death, particularly, the statusquo in China is more serious than other countries. Although considerable progress havebeen made in improving surgery, radiation and chemotherapy as well as other treatments ofliver cancer, the5-years survival rate of liver cancer is still unsatisfactory.With the advance of cancer research, a number of studies have pointed out that theexistence of cancer stem cells (CSCs) is the key for tumor recurrence and metastasis. Toidentify the maintaining conditions of the CSCs and then to develop targeted treatmentshave become a promising direction of oncology researches. Thus, most of scientists haveharbored a keen hope on liver CSCs research.Unlike melanoma and breast cancer which possess relatively specific CSCs markers,even though researchers have established a lot of liver CSCs markers such as CD133,EpCAM, CD90, CD13,CD24and so on, these markers were difficult to approvingly repeatin different laboratories. It is also partly give the reason for arising of so many markerscontinuously. Actually, under the surface flourish, so many markers themselves have madeplenty of confusion and split our focus on the further mechanism research as well.No matter what kind of model has been chosen, the requirement for maintainingpluripotency is the common premise for all of the liver CSCs. Therefore, based on thesimilarity of CSCs to embryonic stem cells (ESCs), we selected Nanog, the importanttranscriptional factor for ESCs self-renewal maintaining, as a novel liver CSCs marker. Inthis study, we focus on the biological characteristic and self-renewal mechanism of Nanogpositive liver CSCs, and discuss the differences and replationships between Nanog and theother markers. Additionally, we move forward to investigate the phenomena andmechanisms of these liver CSCs markers’ interconversion.Methods and Results:1. Nanog could be used as a novel molecular marker of liver CSCs.(1) Nanogcould be used as a marker for the prognosis of HCC. Western Blot analysis showed that Nanog was absent in healthy liver tissues and was higher in HCC tissues than their pairedsurrounding non-HCC tissues; IHC results showed that the expression level of Nanog wassignificantly associated with the recurrence and vascular invasion of HCC. Kaplan-Meier’ssurvival analysis showed that the expression of Nanog in HCC significantly correlated withoverall and disease-free survival.(2) Successful construction of the Nanog promoterreporter system. We constructed a lentiviral vector containing human Nanog promoter todrive the expression of the green fluorescent protein (GFP) reporter gene, and verified thatthe intensity of GFP fluorescence could stand for the expression level of Nanog.(3) Nanogpositive cells possess the stronger ability of self-renewal than Nanog negative counterpart.We discovered that Nanog positive cells have a much powerful ability in sphere formation,clone formation, tumorigenesis and multi-directional differentiation capacity than Nanognegative cells.(4) Nanog positive cells were high metastatic, drug resistant and slow-cycled.Transwell experiments showed that Nanog positive cells have stronger invasion andmigration ability; Western Blot analysis showed Nanog positive cells possess EMTcharacteristic; In vivo translocation experiment showed that Nanog positive cells possesshigh metastatic ability; We also found that Nanog positive cells strongly resist tochemotherapy drugs Sorafenib and cisplatin, and highly expressed of drug resistant genes;By immunofluorescence we showed that Nanog positive cell had a low Ki67positive cellsproportion. Consistently, a single cell proliferation experiment showed that Nanog positivecell cycled slowly.2. Clarify the self-renewal mechanism of Nanog positive liver CSCs.(1) Nanog wasnecessary for self-renewal of live CSCs. Nanog knockdown could significantly reduce thesphere formation, clone formation and migration ability of Nanog positive cells, which alsolead to down-regulation of stemness genes including Oct4and Sox2and up-regulation ofmature hepatocyte markers such as AFP, CK8, CK18, and TTR. Nanog overexpressioncould significantly improve the ability of tumorigenesis and migration of Nanog negativecells.(2) IGF signaling pathway was activated in Nanog positive cells. Microarray analysisshowed that multiple IGF signaling pathway molecules were unregulated in Nanog positivecells, and this was further verified by qRT-PCR showed that increased expression of IGF2and its receptor IGF1R in Nanog positive cells.(3) Nanog could regulate the expression ofIGF1R. Western boltting showed that interfere with the expression of Nanog could reduce the level of IGF1R in Nanog positive cells, and consistently, overexpression of Nanog couldincrease the level of IGF1R in Nanog negative cells. Furthermore, ChIP experimentdemonstrated that Nanog could bind to the promoter of IGF1R, which suggested that Nanogmight regulate the expression of IGF1R in transcriptional level.(4) IGF signaling couldregulate the self-renewal ability and the expression of Nanog. Treatment with IGF1Rinhibitors PPP and AEW541could significantly reduce the expression of Nanog and thesphere formation ability of Nanog positive cells.3. Clarify the profile and correlation of six different kinds of liver CSCs in HCC.(1) Clarify the expressional pattern of six different kinds of liver CSCs. By means of FACS,we tested the positive rate of most kinds of liver CSCs markers, including CD133, EpCAM,CD90, CD24, CD13and Nanog, in3kinds of liver cancer cell lines and5kinds of livercancer primary cells. The results showed that CD13and CD24are highly expressed in mostof tested cells, and the expression pattern of CD133and EpCAM are heterogeneous amongthem, and the percentage of Nanog positive cells was held steady at about10percent, andCD90is barely expressed in tested cells except SMCC-7721.(2) The eight tested livercancer cells can be clearly classified into two groups based on their self-renewal ability.These cells exhibited significant differences in their clone formation, sphere formation andtumorigenicity abilities, so that could be clear divided to two groups.(3) The expressionlevel of CD133and EpCAM could distinguish the two classes of cells. By comparing theexpression level of6markers in both high and low self-renewal cells, we found that theexpression level of CD133and EpCAM were significantly higher in cells with highself-renewal ability.(4) Establishment and application of the method that could co-label for6liver CSCs markers simultaneously. Six different and diacritical kinds of fluorescent, eachstanding for one of liver CSCs markers, were chosen and the compensation between each ofthem were set up by using the Compensation beads from BD Biosciences. Then the settingswere taken used to test the expression pattern of6CSCs markers in8liver cancer cells.(5)There were only existence of limit combinations of6CSCs markers in tested liver cancercells, and distribution map of6CSCs markers in HCC were set up basing on the above data.According to the expression pattern of6CSCs markers, we could separate the global cellpopulation into64subpopulations, and further analysis showed that the major population of8tested liver cancer cells were only distributed in limited subpopulation of them. In advance, by analyzing of the overlap and inclusion relation between every two markers, weestablished the distribution outline of6CSCs markers in HCC.4. Set up a hierarchy differentiation model of HCC based on the expressionpattern of Nanog and CD133.(1) Analysis showed that CD133was the best marker, inabundant biomarkers, for distinguishing the self-renewal ability of tested liver cancer cells.The expression pattern of6CSCs markers in high and low self-renewal liver cancer cellsshowed that CD133was the best marker to distinguish the two groups.(2) Nanog was betterreferential marker than CD133in indication of self-renewal ability. Our data suggested thatNanog positive cells, no matter CD133positive or not, had strong clone formation andsphere formation abilities.(3) Set up a hierarchy differentiation model of HCC based on theexpression pattern of Nanog and CD133. By global and single-cell differentiation tests, wefound that Nanog marked cells unidirectional differentiation from high to low and then tonegative, while CD133labeled cells were interchangeable among the three subpopulation.With further sphere formation ability tests, we concluded that the differentiation direction inthis model was: NanoghighCD133low/neg, NanoghighCD133high, NanoglowCD133pos/neg,NanognegCD133pos, and NanognegCD133neg.5. Illustrated the self-transformation of every markers’ positive and negativesubpopulations, and the interchange among the different liver CSCs markers.(1) LiverCSCs markers were spontaneous self-transformation. The negative and positivesubpopulations of every liver CSCs markers were sorted by FACS, and following by normalor single-cell culture. After days of differentiation, the expressional pattern of the specificmarker were retested. Our results showed that self-transformation of CD133, CD13andCD24positive and negative subpopulations were more obvious among the six markers.CD90negative cells barely changed to CD90positive cells. Interestingly, EpCAM positiveand negative cells were hardly mutually transform in Huh7cells, whereas EpCAM negativecells could irreversibly turn into EpCAM positive cells in T1224cells. And as expected,Nanog postive cells could unidirectional differentiated to Nanog negative cells.(2) Thetransformation of liver CSCs markers are not dependent on the cell division. G0/G1arrestinduced by4mM sodium butyrate treatment could not block the self-transformation ofmarkers’ positive and negative subpopulations.(3) There were interchangeable among the different liver CSCs markers. The6CSCs markers were pairwise grouped into15combinations. And two markers within every combination were co-labeled, gated and sortedby FACS, following by in vitro cultured and retested the markers’ interchange. We foundthat all of surface markers can mutual transformation except for CD90. We also found thatNanog positive cells could generate all of the rest markers positive cells, whereas Nanognegative cells, no matter any one of other markers positive expressed or not, could notconvert to Nanog positive cells under normal culture condition.(4) By summarizing thetransformation data of the various markers in Huh7cells between themselves and each other,we mapped the transformation profile of Huh7cells.6. Illuminated the conditions and mechanisms of Nanog negative cells in vivodedifferentiation.(1) Nanog negative cells could transform to Nanog positive cells invivo. We could detect obvious GFP signal in Nanog negative cells derived tumors. ThenNanog positive and negative cells were isolated from these tumors, and reanalysis of theirstumorigenicity, clone formation and sphere formation abilities. Our data showed that theNanog positive cells source from Nanog negative cells’ dedifferentiation possessedcharacteristic of liver CSCs.(2) Matrigel could be used to induce the transformation fromNanog negative cells to Nanog Positive cells. Nanog negative cells cultured with Matrigelin vitro, could produce the Nanog positive cells, which had stronger ability of sphereformation than corresponding negative cells.(3) Dedifferentiate to Nanog positive cell wasa crucial event for Nanog negative cells’ tumorigenesis. We constructed a lentiviral vectorLv-NTPF which carrier a suicide gene tk under controlling of Nanog promoter, and acontrol vector Lv-NGPF which carrier GFP gene. Western Blot and FACS proved that thissystem could effectively rid of the Nanog positive cells in the whole population. Onceelimination, the abilities of cells’ clone formation, sphere formation and tumorigenesis weresignificantly down-regulated. In vivo experiment showed that cells infected by Lv-NTGFwere difficult to initiate tumor when supplied with a substrate of TK gene GCV, and itsgrowth could be restored upon GCV withdraw.(4) Laminin, matrix component of Matrigel,played an important role in Nanog negative cells’ dedifferentiation. Tumorigenesisexperiment showed that Matrigel and growth-factors reduced Matrigel had the samecontributing to tumorigenesis, which indicated that components responsible for inducingdedifferentiation of Nanog negative cells should belong to the shared matrix components of Matrigel. Then collagen I and laminin-1, two important matrix components of Matrigel,were selected as a supporting condition to culture Nanog negative cell in vitro. Our resultsshowed that Nanog positive cells were only observed after continuously cultured and serialpassaged in the specific condition with laminin-1.(5) Erk/STAT3signaling pathway may beinvolved in the dedifferentiation of Nanog negative cells. Western Blot analysis showed thatthe phosphorylation level of Erk was higher in Nanog negative cells than Nanog positivecells, and consistently, the phosphorylation level was increased after Nanog positive cellsdifferentiation. By contrast, the activity status of STAT3was just exact opposite. WesternBlot also demonstrated that Matrigel and laminin could induce Nanog negative cells’ Erkand STAT3phosphorylation status chang to the same state as Nanog positive cells.Conclusions:1. Nanog can served as a novel biomarker of liver CSCs. In addition, Nanog and IGFsignal pathway can compose a positive feedback loop to regulate the self-renewal of liverCSCs.2. Nanog and CD133are excellent markers to indicate and distinguish the self-renewalability of HCC. Basing on the expression patterns of Nanog and CD133, we could set up ahierarchy differentiation model in HCC, and Nanog is at the relative apex of this model.3. Nanog negative cells could dedifferentiated to Nanog positive cells in vivo. WhileMatrigel could simulate the process in vitro. Laminin, a component of Matrigel, palys animportant role in this process. And the dedifferentiation mechanism may involve inErk/STAT3signals.To sum up, in this study we summary and classify the major liver CSCs markers,which could enable people to understand and appreciate the concept of liver CSCsprofoundly and scientificly. This work has a certain theoretical significance. Moreover, wealso clarify the self-renewal mechanism of Nanog positive cells and the in vivodedifferentiation mechanism of Nanog negative cells, which have a great significance fordevelopment of targeted cancer therapy drugs and clinical application prospect.