Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability

Accumulating evidence supports the concept that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. They are also considered as an attractive target for advanced cancer therapy. Using a sphere culture method that favors the growth of self-renewal cells, we have isolated sphere-forming cells (SFCs) from cervical cancer cell lines HeLa and SiHa. HeLa-SFCs were resistant to multiple chemotherapeutic drugs and were more tumorigenic, as evidenced by the growth of tumors following injection of immunodeficient mice with 1 × 104 cells, compared with 1 × 106 parental HeLa cells required to grow tumors of similar size in the same time frame. These cells showed an expression pattern of CD44high/CD24low that resembles the CSC surface biomarker of breast cancer. We further demonstrated that HeLa-SFCs expressed a higher level (6.9-fold) of the human papillomavirus oncogene E6, compared with that of parental HeLa cells. Gene silencing of E6 with a lentiviral-short-hairpin RNA (shRNA) profoundly inhibited HeLa-SFC sphere formation and cell growth. The inhibition of cell growth was even greater than that for sphere formation after E6 silence, suggesting that the loss of self-renewing ability may be more important. We then measured the expression of self-renewal genes, transformation growth factor-beta (TGF-β) and leukemia-inhibitory factor (LIF), in shRNA-transduced HeLa-SFCs and found that expression of all three TGF-β isoforms was significantly downregulated while LIF remained unchanged. Expression of the Ras gene (a downstream component of TGF-β) was also markedly decreased, suggesting that the growth-inhibitory effect could be via the TGF-β pathway. The above data indicate RNA interference-based therapy may offer a new approach for CSC-targeted cancer therapy.