The Transcription Factor SALL4Regulates Stenrness Of CD133-positive Colorectal Cancer

Colorectal cancer (CRC) is among the three leading causes of cancer-related deathsworldwide. Long-term survival can be achieved only if cancer is completely extirpatedby surgery, particularly in the early, node-negative stage of disease. But in advanceddisease nearly50%of patients occur recurrence or metastasis. Therefore, there is anurgent need to seek for the mechanism。Accumulating evidence suggests that a subpopulation of tumor cells with distinctstem-like properties is responsible for tumor initiation, invasive growth, and metastasisformation. This population is defined as cancer stem cells (CSCs).Recent research showthe existence of CSCs in various types of tumors，including colorectal cancer. Currently,there are several factors reported to be putative CSCs of CRC: CD133, CD44, Lgr5,ALDH1A1, et, al. Targeting these factors will be useful for preventing tumor growth byaltering the balance between self-renewal and differentiation of cancer stem cells.The transcription factor SALL4, known to regulate stemness in embryonic andhematopoietic stem cells，similar to OCT4, SOX2and NANOG. Recent studies havefound sall4playing a critical role in solid cancers，such as：liver cancer，gastric cancerand endometrial cancer. In this study, we aimed to investigate the transcriptionalprograms regulating the stemness of cancer stem cells in colorectal cancers.The study consists of two parts:(1) Expression of SALL4in CRCs and the re-lationship with clinicopathological characteristics；(2) SALL4regulates stemness ofCD133-positive colorectal cancer.Part1. Expression of SALL4in CRCs and the relationship withclinicopathological characteristicsAs reported, the transcription factor SALL4has been considered as a valuablebiomarker of many cancer cells. Firstly, we investigated the expression of SALL4inpatients of colorectal cancer by Immunohistochemistry. We constructed a panel of tissuemicroarrays consisting of155surgically resected specimens of primary colorectalcancers and matched, archived, non-neoplastic colorectal specimens obtained from theEastern Hepatobiliary Surgery Hospital. To analyze the clinical relevance of SALL4reactivation in colorectal cancer, we carried out a clinicopathological analysis. Theanalysis showed that SALL4is positive in part of the cancer tissues,but keep silent in non-neoplastic tissues.Patients with high expression of SALL4had a worse prognosisthan patients with a low level of SALL4expression. We further examine the expressionof CD133in the same microarray cohort. We validated the strong positive correlationbetween SALL4and stem cell markers CD133,which suggesting the role of SALL4incolorectal cancers was involved with CD133.We further performed reversetranscriptase-PCR (RT-PCR) on50CRCs tissues. The results also showed that a positivecorrelation between SALL4and CD133.Part2. SALL4regulates the stemness in CD133+colorectalcancer stem cellsWe detect the expression of SALL4and CD133in three colon cancer cell linesHCT116, RKO and sw620. In the level of protein and gene, both of the results showedthe similar trend. In addition, Immunofluorescence analysis conformed mostly CD133+HCT116cells performing high expression of SALL4. By flow cytometry sorting,HCT166cells are divided into two subgroups: CD133+and CD133-cells,The geneexpression of SALL4examined by RT-PCR in CD133+group is higher than that ofCD133-HCT116cells.In order to verify the regulation of the stemness in CD133+colorectal cancer stemcells by SALL4, RKO,a cell line expressing low CD133, used as theSALL4-overexpressing model, after transfection, the stem cell markers CD133and Lgr5were raised, colony-forming ability and tumorigenicity is also significantly enhanced.wealso employ short hairpin RNAs (shRNAs) specifically targeting SALL4in HCT116cells. We found that SALL4knockdown caused a decline in cancer stem cell phenotype.Results:1. Transcription factor SALL4is highly expressed in CRCs, and indicates worseprognosis;2. There is an opsitive correlation between SALL4and stem cell markers CD133inCRCs.3. SALL4can regulate the “stemness” of colorecal cancer stem cells.