ATXN1Promotes Invasion Of Medulloblastoma And ALDH1Enhances Invasion Of ESCC: Clinical Significance And Molecular Mechanisms

Recently, the cancer stem cell （CSC） hypothesis suggests that tumors are organized intoa hierarchy in which only a rare clonal population of cells, termed CSCs or tumor initiatingcells （TICs）, have the ability to initiate, proliferate, and maintain tumor growth. Emerging ofCSCs hypothesis provides us a new way insight into the mechanism underlining tumorinitiation and progression. This article included two parts as follows:PartⅠ: The role and correlated molecular mechanisms of ATXN1in stemness andinvasion of medulloblastoma.Medulloblastoma is the most common malignant brain tumor in children. Despite recentimprovements in cure rates, survivors suffer from serious therapy-related side-effects. Manysurviving children have long-term cognitive and/or neuroendocrine adverse effects. Todemonstrate the existence of medulloblastoma TICs （MTICs） and explore their malignantbehavior, we established a method of sphere forming culture to gain the tumor spheres frommedulloblastoma cell lines. Tumor sphere cells possessed multi-potent differentiation ability,highly invasion and tumorigenicity, indicating that tumor spheres enriched MTICs.Ataxin1（ATXN1） is a polyglutamine protein of unknown function, whose mutant formcauses spinocerebellar ataxia type1（SCA1）. ATXN1over-expressed in both lung cancer andgastric cancer stem cells, was also found over-expressed in MTICs. Then, the roles andcorrelated molecular mechanisms of ATXN1in stemness and invasion of medulloblastomawere explored. Main methods, results and conclusions of this part as follows:1. The primary culture and establishment of medulloblastoma cell line from Chineseadult patients. For better comprehend the biologic traits of medulloblastoma and satisfy theneeds of experimental research, five cases of medulloblastoma specimen were collected andproceed cell culture. Fortunately, one primary medulloblastoma cell line was successfully established. Light microscopy, electro microscopy, immunohistochemistry, flow cytometry,karyotype analysis and tumor implantation experiments were carried out to characterize thebiological behaviors.2. Tumor spheres formed from medulloblastoma possess stem cell properties.（1）medulloblastoma cell lines of Daoy, ons76and D283could grow as tumor sphere in serumfree conditioned medium.（2） Compared with monolayer cells （MN）, sphere cells （SC） highlyexpressed stemness-related genes Bmi1, Nanog, Nestin, Oct4and Sox2. Invasion-relatedgenes MMP2, MMP7, MMP9and MMP13were all over-expressed in SC compared to MN.（3） Immunofluorescence staining results indicated that Daoy-SC with increased-expression ofstemness-related protein Nestin, Sox2and CD133, decreased expression of differentiatedmarker GFAP, MBP and βⅢ-tubulin.（4） Cultured Daoy-SC in medium supplemented with10%FBS, the cells could be induced to express GFAP, MBP and βⅢ-tubulin positive cells.（5）Transwell matrigel invasion assay was performed to assess the invasion capacity, we foundthat indicating Daoy-SC possess higher invasion capacity compare to Daoy-MN.（6）Xenograft assay showed that Daoy-SC were highly tumorigenic. Those data suggested thatsphere cells possess stem cell properties.3. Our results demonstrated that ATXN1plays an important role in immigration, invasion,self-renewal and tumorigenesis in medulloblastoma.（1） ATXN1was found highly expressedin MTICs by real time PCR and Western blot.（2） Silenced ATXN1expression inmedulloblastoma cells by shRNA, the abilities of sphere formation, invasion andtumorigenesis were impaired.（3） Over-expressing ATXN1, the abilities of invasion andtumorigenesis of D283cells were increased.4. ATXN1expression has important clinical significance in patients withmedulloblastoma. Immunohistochemistry （IHC） was used to detect the expression of ATXN1in262specimens of medulloblastoma, the result showed that the expression of ATXN1wascorrelated with histological subtypes and molecular subgroups. Patients with lower ATXN1expression had longer overall survival time than higher ones （P=0.0023）, indicating thatATXN1is an indicator of poor prognosis in medulloblastoma.5. The possible molecular mechanisms of ATXN1in invasion and self-renewal abilitiesof medulloblastoma cells were explored.（1） Silenced ATXN1expression in Daoy cells byshRNA, the expression of stemness-related gene Nanog and invasion-related gene MMP2 were down-regulated, meanwhile, Gli1, the key molecular of Hh signal pathway, wasdown-regulated significantly.（2） Overexpressed ATXN1in D283cells, Nanog, MMP2andGli1were all up-regulated significantly.（3） The Shh ligand and KAAD-cyclopamine couldstimulate or inhibit the expression of Gli1and ATXN1in Daoy cells. These results imply thepresence of a positive loop between ATXN1and Gli1.（4） Silencing Gli1expression bysiRNA resulted in decreased expression of Nanog and Gli1, but expression of ATXN1was notinfluenced, so we supposed ATXN1might be an upstream factor of Gli1to exert effect.PartⅡ: ALDH1plays important roles in esophageal squamous cell carcinoma（ESCC） stemness maintenance, invasion and metastasis.ESCC is one of the most frequent fatal malignancies. The five year survival rate ofESCCs after surgery and chemotherapy remains low due to highly invasive and metastaticnature of ESCC. Recent studies suggest that TICs are responsible for invasion andmetastasis of many tumor types. Therefore the biomarkers related to invasion and metastasisof TICs need to be identified in ESCC.Aldehyde dehydrogenase1（ALDH1） is a TIC-associated protein in various malignanttumors and its level correlates with the patient outcome. We hypothesize that ALDH1mayalso be used to detect and enrich TICs and the level of ALDH1^highcells in tumor may predictthe prognosis of human ESCC. Main methods, results and conclusions of this part as follows:1. ALDH1^highESCC cells exhibited TICs properties. We examined the proportion ofALDH1^highcells contained in ESCC cell line EC109, and found that the sorted ALDH1^highcells possessed higher capabilities of tumor sphere formation as well as colonies formationas compared to ALDH1lowones. The ALDH1^highcells also showed increased expression ofthe stemness genes Sox2and Bmi1and higher tumorgenecity compred to ALDH1lowcells.2. ALDH1^highcells possessed highly invasive and metastatic capabilities with EMTphenotype. Matrigel invasion assay indicated that ALDH1^highcells had a greaterinvasiveness as compared to ALDH1lowones. Moreover, the ALDH1^highcells formed lungmetastasis in five of six （5/6） mice, whereas no metastasis was found by the ALDH1lowones.Gene expression analysis further showed that the ALDH1^highcells expressed higher levelsof matrix metalloproteinase （MMP）-2,-7,-9and vimentin, and lower level of E-cadherin,indicating their highly invasive and metastatic capabilities related to EMT phenotype.3. ALDH1expression was correlated with esophageal dysplasia and ESCC malignancy. ALDH1was not detected in normal esophageal epithelia, but detectable in the dysplasticbasal cells to some extents. Furthermore, the cytoplasmic ALDH1was elevated in theexpression levels along with the increasing grades of dysplasia and the formation ofcarcinoma in situ. The expression of ALDH1was positively correlated with histologicalgrade （P=0.004）, invasion depth （P=0.000）, lymph node metastasis （P=0.002） andUICC stage （P=0.000） of ESCCs.4. ALDH1is associated with ESCC outcome and can be used as a biomarker forpatient prognosis.