Sox9 Regulates Sell-Renewal And Tumorigenicity By Promoting Symmetrical Cell Division Of Cancer Stem Cells In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the common malignant tumors occurred in the digestive system. HCC is the fifth most prevalent tumor and the third lethal cause of cancer-related deaths in the worldwide. Although surgery and radio frequency ablation (RFA) are the best option for small tumors at early stage of HCC patients, the tumor recurrence and resistance to chemotherapy and radiotherapy result in poor outcome of HCC. Therefore, it will provide great significance to further study on the mechanism about initiation, development and rescue of HCC. And it’s urgent to discover the new molecular markers which can be used in the early diagnosis, preventment and individual therapy of HCC, thus eventually improve the life quality of the patients.Studies have demonstrated that a subpopulation of cancer cells, often referred to as cancer stem cells (CSCs) or tumor initiating cells (TICs), are capable of extensive proliferation, self-renewal, and the increased frequency of tumor formation. The concept of CSCs has significant clinical implications:CSCs have been shown to be more resistant to chemotherapy and radiotherapy; CSCs have the properties of EMT, metastasis and tumor relapse after surgery. Therefore, understanding common mechanisms in self-renewal of CSCs will enable further research to focus efforts on therapeutic targets which may be most useful in developing new approaches of treatment. Our previous study have identified transcription factor Nanog as a novel marker for liver CSCs and demonstrated that Nanog could play a crucial role in regulating self-renewal of liver CSCs [Shan et al., Hepatology 201656(3):1004-1014l Nevertheless, the key components and molecular mechanisms contributing to biology of liver CSCs are largely unknown.The Sox9 (Sex Determining Region Y-Box 9) is well-established regulators of cell fate decisions during development. Sox9, located at q24.3-25.1 at chromosome 17, length about 3934bp and contained two introns, is the first reported containing introns of Sox gene. During tumorigenesis, the Sox E group member Sox9 was upregulated in a number of tumors and as an oncogene plays an essential for cancer progression, including HCC. In addition, high levels of Sox9 expression enhance the tumorigenic and mtastasis-seeding abilities of human breast cancer cells in a transplant model, and EMT confers on the cells many of the properties associated with cancer stem cells, respectively. A mouse model of basal cell carcinoma has revealed that Sox9 is required for CSC self-renewal and the maintenance of balance between symmetrical cell division (SCD) and asymmetrical cell division (ACD) during skin tumorigenesis. Furthermore, mice with constitutively-active Notch develop hepatic tumors that have increased Sox9 expression levels. Consistently, our previous results have found that Notch signaling activity is essential for the maintenance of stem characteristics in liver CSCs.In view of all results above, we wonder whether Sox9 play an important role in maintain liver CSCs self-renewal? Whether symmetrical division affects Liver CSCs self-renewal and tumorigenicity? Whether symmetric division of liver CSCs populations relies on Sox9 regulates liver CSCs self-renewal and tumorigenicity? Therefore, we aimed to determine whether Sox9-directed SCD of liver CSCs could modulate the proliferation and self-renewal capacity of human liver cancer cells.1. Sox9 was elevated in self-renewing liver cancer cells and regulates proliferation and sternness properties in liver CSCs.We first used immunoblotting and immunofluorescence assays to determine the pattern of Sox9 expression in freshly-purified liver CSCs and differentiated cancer cells obtained using our established PNanog-GFP CSC-expanding system. Next, primary tumors derived from liver CSCs or non-CSCs grown in NOD/SCID mice were removed and examined. We obtained freshly and paraffin-emmbedded HCC tissues and surrounding nontumor hepatic tissues from 175 HCC patients who had undergone radical resection between 2005 and 2011. Sox9 was significantly highly expressed in HCC tumors, while being almost undetectable in non-cancerous and healthy liver tissues by western blot and IHC. Kaplan-Meier analysis indicated that high Sox9 expression in HCC was significantly correlated with reduced overall survival.We next addressed the importance of Sox9 in liver tumorigenesis by assessing CSC proliferation following silencing of Sox9 expression in the HCC cell lines. Four short hairpin RNAs targeting Sox9 were designed and produced. Proliferation, cell cycle and Ki-67 staining analysis revealed that Sox9-knockdown (Sox9-KD) significantly reduced cell growth. Stem cell transcription factor and differentiation markers analysis determined that Sox9-KD cells exhibited caused significant downregulation, and a significant upregulation of differentiation marker levels.We next sorted liver CSCs cell populations by FACS to examine the role of Sox9 in self-renewal and tumorigenicity in vitro and in vivo. Sox9-KD CSCs exhibited deceased capacities for expansion and self-renewal as determined by clonogenic and mammosphere structures assays in which cells were seeded. Next, we observe that Sox9-KD CSCs required more cells and longer incubation times in vivo to generate tumors to equivalent size to those formed by purified control CSCs. To further tested Sox9 levels are critical for stem/progenitor characteristics, we ectopically expressed Sox9 in non-CSCs cells, and then determined whether ectopically expressed Sox9 increases non-CSCs self-renewal and tumorigenicity capacity. These results demonstrate that Sox9 is an important regulator of the expansion and tumor progression in liver CSCs.2. An increased symmetrical division in Liver CSCs.Owing to the critical role of regulating the mode of cell division in CSCs, and we tested the cell division of liver CSCs by pulse-chase BrdU labelling and paired-cell assays method. Compared with differentiating of liver CSCs in DMEM with 10% FBS, liver CSCs were cultivated in sphere culture medium (SCM) that were maintaining for stem-like populations and exhibited a greater tumour-initiating ability using a serial transplantation assay. Gene expression analysis indicated that the expression of stemness genes was increased in cells cultivated in sphere culture medium. We further confirmed liver CSCs reduced sphere-forming capability, clonogenicity, tumour initiation capability, SCD of CSCs and increased Numb expression when blockade Notch pathway which reduced stem-like populations. These results confirmed an increase SCD in liver CSCs populations.3. Repression of Numb by Sox9 Promotes Stemness and SCD in Liver CSCs. We next examined whether Sox9 could influence cell division of liver CSCs to promote tumor initiation using paired-cell assays. We firstly investigated relationship between Sox9 and BrdU-label by paired-cell assay, and results showed that Sox9 was frequently co-localized with pre-labelled BrdU. Additionally, the knockdown of Sox9 in CSCs from HCC cell lines reduced SCD. Moreover, we also found that the inhibition of Notch by the y-secretase inhibitor in combination with overexpression of Sox9 was restored liver CSCs tumorigenicity and SCD. These date suggest that endogenous Sox9 promotes liver CSCs self-renewal and expansion of stem/progenitor cell populations by regulating the balance of SCD versus ACD.In the majority of systems, Numb acts as a Notch antagonist, although in at least one context, Numb can act synergistically with Notch. A relatively low level of Numb was detected in liver CSCs, while increased Numb levels were present following differentiation of liver CSCs populations in vitro. Silencing or ectopic Sox9 expression could regulate Numb protein expression. Meanwhile, we also found that overexpression Numb was significantly decreased Sox9 expression. Additionally, ectopic Sox9 expression downregulated Numb levels in the presence of the Notch pathway inhibitor. These results suggest that Sox9 represses Notch signaling antagonist-Numb expression to enhance stemness and SCD. Finally, we examined the clinical significance of the Sox9-Numb loop in HCC patients. An inverse correlation between Sox9 and Numb was observed, and the Sox9High NumbLow profile was an indicator of poor prognosisThe following conclusions were reported according to the above study.1. Sox9 was elevated in self-renewing liver cancer cells and correlates with poor prognosis in HCC Patients.2. Sox9 plays an important role in liver CSCs self-renewal and tumorigenicity capacity.3. Symmetric division of liver CSCs populations relies on Sox9.4. Repression of Numb by Sox9 promotes stemness and SCD in liver CSCs. Clinical analyses revealed that Sox9Hlgh NumbLow profile is associated with poor prognosis in human HCC patients.