Study On Loss Of Insulin-like Growth Factor 2 Imprinting In Cancer Stem Cells And Its Mechanisms

The discovery of cancer stem cells(CSCs) is a landmark in cancer research that has broad potential applications for targeted tumor therapy. Tumor cells contain both CSCs and non-stem tumor cells(n SCCs), CSCs are a small subgroup of cells residing in a heterogeneous population of tumor cells. CSCs, which usually comprise <1% of all cells in a given tumor, are thought to drive tumor progression, invasion, migration, metastasis, and drug-resistance. Cancer stem cells have been isolated from many kinds of malignant tumors.Conventional cancer therapies may eliminate most of the tumor mass, but a small population of CSCs with the potential to repopulate the tumor may escape destruction and survive the therapy. Since successful eradication of a malignancy may require the elimination of the CSCs, it would be ideal to find a therapy that can specifically target and kill CSCs.The growth hormone/insulin-growth factor(IGF) axis plays an important role in regulating self-renewal of cancer stem cells. The IGF pathway is frequentlyactivated in a variety of cancers. By stimulating the PI3-K and MAPK cascade pathways, IGF-I and IGF-II promote cell proliferation and induce resistance to chemotherapeutic agents, radiation, and targeted therapies. IGF2 is maternally imprinted in most normal tissues, with only the paternal allele being expressed. In many tumors, however, this imprinting is lost, leading to biallelic expression of the gene. Over-production of the growth factor promotes the malignant behavior of tumor cells through enhanced cell growth and CSC self-renewal, and loss of IGF2 imprinting(LOI) is associated with tumor initiation. Moreover, IGF2-overexpressing tumors frequently display loss of PTEN, and they are often highly proliferative, exhibiting strong staining for phospho-Akt. But there are few studies on Molecular mechanisms underlying aberrant genomic imprinting of IGF2 LOI in the "stem" maintenance of cancer stem cells. Objective:1. We aimed to study loss of IGF2 imprinting in CSCs, compare the differences of CSCs and n SCCs on IGF2 imprinting;2. We aimed to study the roles of IGF2 LOI plays in maintance stem cell characteristics, self-renewal and resistance to chemo/radiotherapies;3. We aimed to reveal epigenetic mechanisms of IGF2 LOI in CSCs. Methods:1. Flow cytometry sorting technology was used to isolate CSCs and non-CSCs from solid tumor cell lines by using an R-PE conjugated monoclonal antibody against CD133.2. Serum free floating culture assay was used for enrichment of CSCs3. Flow cytometry analysis of CSCs marker, CSCs sphere culturing and immunofluorescence analysis were used in identification of CSCs.4. Sequencing method was used to determine the status of IGF2 imprinting of CSCs.5. q RT-PCR method was used to examine gene expression of IGF2 in CSCs; Western blotting was used to examine protein expression of IGF2 in CSCs.6. Soft-agar clonogenic assay was used to examine the tumorigenicity of the cultured CSCs.7. WST-1 cell proliferation assays was used to assess the chemotherapy and radiotherapy sensitivity of CSCs.8. Chromosome conformation capture(3C) was used to detect intrachromosomal looping of CSCs.9. Using chromatin immunoprecipitation(Ch IP) to determine whether the epigenetic suppressive mark H3K27 methylation was altered in CSCs in an association with IGF2 LOI.10. We used si RNAs to knock down IGF2 in CSCs, and cell proliferation array, cell cycle and apoptosis analysis and cell migration and invasion array were used to further examine the role of IGF2 in CSCs, Results:1. Using an R-PE conjugated monoclonal antibody against CD133,CSCs and non-CSCs were isolated from solid tumor cell lines by flow cytometry sorting technology.The CSCs were cultured in nonadhesive plates in cancer sphere medium.We found that the morphology of sphere-forming-like cells after several passages maintained the same characteristics as that of the first passage.2. Using immunofluorescence, we found that CSC marker CD133 and ALDH continued to be positive expressed on the cell surface of 6 kinds of CSCs.3. Using flow cytometry to analysis the expression of CSCs marker CD133+, we found that more than 90% of the spheroid cells stained positive for CD133.4. Using sequencing method to determine the status of IGF2 imprinting of CSCs, we found IGF2 LOI in CSCs, even when the stem cells were derived from a cell line in which the general population of cells maintain IGF2 imprinting.5. Using q RT-PCR method to examine gene expression of IGF2 in CSCs,we found that IGF2 was significantly upregulated in CSCs as compared with their non-CSC counterparts; Using western blotting to examine protein expression of IGF2 in CSCs, we found that IGF2 was significantly upregulated in CSCs as compared with their non-CSC counterparts.6. Using Soft-agar clonogenic assay to examine the tumorigenicity of the cultured CSCs,we found CSCs with IGF2 upregulation exhibited a significantly greater ability to form colonies than did the non-CSC cells in all cell lines tested.7. Using WST-1 cell proliferation assays to assess the chemotherapy and radiotherapy sensitivity of CSCs, we found CSCs exhibited greater chemo/radiotherapy resistance than did the non-CSCs.8. Using Chromosome conformation capture(3C) to detect intrachromosomal looping of CSCs,we found a significantly lower intrachromosomal interaction signal in CSCs than that seen in non-CSCs.9. Using chromatin immunoprecipitation(Ch IP), we examined H3K27 methylation in the IGF2 promoters. We observed a significant decrease in IGF2 promoter H3K27 methylation in CSCs as compared with the non-CSCs.10. We used si RNAs to knock down IGF2 in CSCs to further examine the role of IGF2 in CSCs. After IGF2 knockdown, we found that: a time-dependent inhibition of cell proliferation; an increased percentage of cells in G2/M-phase in parallel with a decrease in G0/G1 phase; an increase in cell apoptosis in CSCs; a reduction in cell migration and invasion; resistance to chemo/radiotherapies. Conclusions:1. Loss of IGF2 imprinting is present in CSCs. IGF2 LOI maybe is a common feature of CSCs.2. IGF2 LOI in CSCs plays an important role in maintance stem cell characteristics of self-renewal and resistance to chemo/radiotherapies.3. IGF2 LOI is assosiated with disappearance of intrachromosomal looping and gene epigenetic modification of promoter H3K27 methylation.