Sox2Regulates The Characteristics Of Cancer Stem-like Cells Derived From Human Gastric Cancer Cells

With the research going further, people have realized that the properties of malignant tumor, such as genesis, development, metastasis and recurrence were amazingly similar to what of the normal stem cell. Stem cells are identified as a class of immature undifferentiated cell populations with self-renewal and differentiation capacity, and reserve the asymmetric replication phenotype. There is now compelling evidence that the bulk of the malignant cells in cancers are generated by rare fractions of self-renewing, multipotent and tumor-initiating cells, termed cancer stem-like cells (CSCs). The last decade has witnessed a transition from a state of assumptions of stem cells in the onset and progression of cancer to the establishment of actual experimental proof of the isolation and identification of CSCs in most kinds of tumors, including leukemia and solid tumors sush as in breast, brain, lung, prostate, pancreas, gastric cancer and others. The CSCs are usually associated with the development of drug resistance, tumor dormancy, minimal residual disease, and disease relapse. However, the behind mechanisms how these properties maintained in CSCs are need to be further elucidated. Therefore, it is an urgent challenge to understand the detail molecular events of these cells from a developmental and evolutionary aspect, and that may be crucial in identifying the aberrant events leading to disease.Objective:To isolate and evaluate the general properties of the GCSCs from human gastric cancer (GC) cell lines SGC-7901, BGC-823, MGC-803, HGC-27and MKN-28; to identify key molecules in maintaining the biological characteristics of these GCSCs, and to develop targeted interventions for inhibiting tumorigenesis induced by GCSCs.Methods:1. FACS sorting based on SP analysis was used to isolate gastric CSCs from five human GC cell lines SGC-7901, BGC-823, MGC-803, HGC-27and MKN-28. The biological differences between SP and NSP cells were identified by analysis of abilities in asymmetric cell division, plating, spheroid colony formation and drug resistance in vitro.2. Gastric floating sphere cells were obtained by culturing SGC-7901and BGC-823cells in serum free medium, and the biological differences between sphere cells (SC) and the adherent cells (AC) were performed by detecting the capability of drug resistance in vitro and tumorigenicity in vivo.3. Real-time RT-PCR was conducted to identify the teranscriptional expressions of SP phenotype-related ABCG2and the other ABC transporters such as MRP1, MRP2and MDR1genes in SP and NSP cells, SC and AC, as well as some potential sternness genes including Sox2, Oct-4, HES1, Krt15and CD44. Next, Western boltting was utilized to examine the protein expression levels of ABCG2between gastric SP and NSP cells and Sox2between gastric SC and AC, respectively.4. Doxorubicin efflux analysis, spheroid colony formation assay and apoptosis analysis in vitro and tumorigenicity in vivo were performed using Sox2siRNA-transfected SC and control cells derived from SGC-7901and BGC-823cells.Results:1. The ratios of SP cells were0.5%for SGC-7901cells and both0.6%for BGC-823and MGC-803cells, respectively, however, there was no SP cells detected in HGC-27and MKN-28cells by SP analysis. Then,5×104SP cells and6x104NSP cells were sorted by FACS from1x107SGC-7901and BGC-823cells, respectively. CD44expression was significantly higher in SGC-7901, BGC-823and MGC-803cells than in HGC-27and MKN-28cells (*P<0.05). SP cells were proliferated in an asymmetric cell division-like manner. In addition, SP cells derived from SGC-7901and BGC-823cells exhibited higher potentials of plating, spheroid colony formation (8.4-,5.0-fold, respectively,**P<0.01) and drug resistance (*P<0.05,**P<0.01) than NSP cells, respectively.2. Only a small portion of SGC-7901and BGC-823cells can survive in serum-free culture condition and form anchorage-independent suspended spherical colonies with the ability of continuous proliferation and self-renew. Furthermore, the cultured sphere cells were found with significantly enhanced capabilities of drug resistance to both doxorubixin and cisplatin in vitro (*P<0.05,**p<0.01) and tumorigenicity in vivo (6.2-,11.9-fold, respectively,*P <0.05,**p<0.01).3. The Real-time RT-PCR revealed that the SP phenotype-related ABCG2transporter expression was little elevated in SGC-7901SP cells and SC than other cells. We also found that MRP2and MDR1were overexpressed in these intrinsic drug resistance cells (SP and SC derived from SGC-7901and BGC-823cells). It was also shown that Oct4, Krt15and CD44, particular Sox2were upregulated in gastric SP cells and SC (*P<0.05,**P<0.01). The results of Western blot suggested identical difference in ABCG2protein between gastric SP and NSP cells; but gastric SC expressed noteworthy enhanced Sox2level than gastric AC.4. Knockdown Sox2with special siRNA obviously reduced spheroid colony formation (1/5.4-,1/6.0-fold, respectively,**P<0.01) and doxorubicin efflux in SGC-7901-SC and BGC-823-SC increased their apoptosis rate in vitro (**P <0.01) and suppressed tumorigenicity in vivo.Conclusions:1. We successfully isolated CSCs from human gastric cancer cell lines SGC-7901 and BGC-823. Both SP cells and cultured sphere cells are enrich with GCSCs. Suggesting that SP sorting and culturing sphere cells in SFM can be used to isolate and characterize the CSCs from GC cell lines.2. ABC transportation proteins ABCG2, MRP2and MDR1are related to SP phenotype and drug resistance. Stem cell markers Oct4, Krt15and CD44, especially Sox2are essential in maintaining gastric CSCs properties.3. Sox2plays a pivotal role in the tumorigenicity of GCSCs and sustaining their stem cell properties and might be a potential target for developing personalized therapy in gastric cancer.