The Role Of SHH Pathway In Gastric Cancer Stem-like Cells

CSCs play an important role in cancer development, this subpopulation within tumor possesses the characteristics of self-renewing capacity, chemoresistance and tumorigenic capacity thus may play a crucial role in the initiation, progression and recurrence of cancer. Examining and manipulating the biochemical pathways involved in those characteristics is one of the best ways of contributing to CSCs, leading to the development of novel drugs and treatment procedures.SHH pathway plays a critical role in many CSCs, such as glioblastoma stem cells, CD34+ leukemic cells and breast CSCs, in addition, it is crucial to the development and homeostasis of gastric gland, abnormal activation of the SHH pathway could result in gastric cancer, however, the role of SHH pathway in gastric CSCs is not clear. SHH pathway in mammalian cells is mediated by ligands Shh. In the absence of Shh, the transmembrane receptor patched (Ptch) inhibits the activity of another transmembrane protein, smoothened (Smo), resulting in inactivation of SHH pathway. Binding of Shh to Ptch abrogates the inhibitory effect of Ptch, and Smo is derepressed, thereby activating transcription factor Gli(Gli1,Gli2 and Gli3). Glil is a strong positive activator of downstream target genes and is itself a transcriptional target of SHH pathway. Therefore, Glil is considered a marker of SHH pathway abnormal activation.In this study, we investigated the possibility that abnormal activation of the SHH pathway maintained the characteristics of gastric CSCs. First, we identified cancer stem-like cells from human gastric cancer cell lines (HGC-27, MGC-803 and MKN-45) using tumorsphere culture. Compared with adherent cells, the floating tumorsphere cells had more self-renewing capacity and chemoresistance. The cells expressing CSCs markers (CD44, CD24 and CD133) were also significantly more in tumorsphere cells than in adherent cells. More importantly, in vivo xenograft studies showed that tumors could be generated with 2×104 tumorsphere cells, which was 100-fold less than those required for tumors seeding by adherent cells. Next, RT-PCR and Western blot showed that the expression levels of Ptch and Gli1 (SHH pathway target genes) were significantly higher in tumorsphere cells than in adherent cells. The results of quantitative real-time PCR were similar to those of RT-PCR and Western blot. Further analysis revealed that SHH pathway blocked by cyclopamine or 5E1 caused a higher reduction in self-renewing capacity of HGC-27 tumorsphere cells than that of adherent cells. We also found that SHH pathway blocking strongly enhanced the efficacy of chemotherapeutic drugs in HGC-27 tumorsphere cells in vitro and in vivo but had no significant effect in adherent cells. Finally, we isolated the tumorspheres from gastric cancer specimen, these cells also had chemoresistance and tumorigenic capacity, and SHH pathway maintained the gastric CSLCs characteristics of tumorsphere cells from primary tumor samples. In conclusion, our data suggested that SHH pathway was essential for maintenance of CSLCs in human gastric cancer.