It is well known that PDX-1 is required for embryologic development of the pancreas and for maintenance of hormone expression in the adult islet35–40
. PDX-1 has also been found to regulate proliferation of islet cells in mice15
as well as proliferation and invasion of pancreatic cancer cells in vitro14–16
. Re-expression of PDX-1 in adult duct cells, under certain conditions such as pancreatectomy and pancreatitis, suggests that PDX-1 may be associated with the regenerative responses that accompany these conditions. It has been demonstrated to be a key regulator of the induction of cell differentiation from non islet cells to insulin-secreting cells41
, especially acinar to ductal cell metaplasia, a common cellular change that may progress to malignancy22
. These studies, together with the finding that PDX-1 is overexpressed in most solid cancers, which is associated with advanced clinical pathological stages and poor prognosis of patients with pancreas cancer, suggests that PDX-1 could be playing a role in oncogenesis 25, 26, 29, 31, 32
. To test the hypothesis that PDX-1 is an oncogene, we utilized techniques similar to those performed on Kras, which is a well known pancreas cancer oncogene that induces malignant cellular transformation when over-expressed as an activating point mutation at codon 12( KrasG12D
)1, 2, 42–44
. Our cumulative data show that over-expression of PDX-1, in addition to affecting cell proliferation and invasion in both benign and malignant human cell lines, induced significant cell transformation by colony formation and promoted tumor growth in vivo
, strongly supporting the role of PDX-1 as a potential oncogene. An opposite view has been mentioned in one study in which PDX-1 was considered as a tumor suppressor gene in human gastric cancer 45
. Further studies are needed to clarify this issue, since most studies showed high level of PDX-1 in gastric cancer specimens46–50
, as opposite to this study showing low expression of PDX-1 in a single human gastric cancer cell line.
To strengthen the evidence of PDX-1’s role in tumorigenesis, we also utilized visualized PDX-1 expression and PDX-1 siRNA knockdown techniques for in vitro
studies in multiple human cell lines. HEK 293 cells are routinely used as a “normal” utility cell to test the function of oncogenic or tumor suppressor genes51
. HPDE cells, which originate from human pancreatic ductal epithelial, have been widely used as tool to investigate the oncogenic property in pancreatic cancer studies 52, 53
. MIA PaCa2 cells and PANC-1 cells are human pancreatic cancer cell lines with low and high endogenous expression of PDX-1, respectively. Consistent results were obtained from different human cell lines, demonstrating reliable approaches used in the study, emphasizing the crucial role of PDX-1 in mediating tumorigenesis. Secondly, visualized PDX-1 expression offered kinetic observation of cellular responses to PDX-1 expression. It was particularly useful in monitoring PDX-1 expression in colony formation of PDX-1-transformed cells. Lastly, RNAi technique was used to validate the role of PDX-1 in regulating cell proliferation and invasion. By knockdown of PDX-1 expression, the effectiveness of PDX-1 induction was accordingly reversed, providing confirmative evidence of PDX-1’s role in cell proliferation and invasion. In combination of three approaches, the study provides reliable evidence to demonstrate that PDX-1 mediates tumorigenesis and further support our previous view that PDX-1 is therapeutic target for pancreatic cancer28
. Using a xenograft human cell-SCID mouse model, this study demonstrates in vivo
evidence that PDX-1 overexpression induces tumor formation and growth. HEK 293 cells harboring PDX-1 expression grew large tumors in all implanted mice, whereas HEK 293 control cells developed only a very small tumor in one of five mice. The results are consistence with other studies using HEK 293 cells to study the function of other oncogenes51, 54
. PDX-1 also promoted MIA PaCa2 cell tumor growth in SCID mice, indicating PDX-1 also has a cumulative effect on tumorigenesis, as MIA PaCa2 control cells have low endogenous PDX-1 expression. These data further support the hypothesis that PDX-1 is an oncogene.
In terms of the mechanism by which PDX-1 is involved in pancreas cancer tumorigenesis, we have shown that overexpression mouse PDX-1 in human pancreas cancer cell lines, as well as PDX-1 shRNA knock down of PDX-1, results in disruption of cell cycle proteins28
. Previous PDX-1 studies have shown dependence on several signal transduction pathways such as those involving Stat322
, and phosphatidylinositol 3-kinase/Akt/mTOR signaling pathways56
. There is great deal of overlap between these transduction cascades and those described in SHH regulation of proliferation in pancreatic cancer57
. The data in this study demonstrate PDX-1 up-regulates expression of Cdk2, cyclin E, and down-regulation of p27, p21 and P53 expression in human PDX-1 overexpressed HEK 293 cells both in vivo
and in vitro
; these data are consistent with our previous observations in other cell lines, addressing the role of PDX-1 on G1 to S transition of cell cycle, emphasizing the critical role of PDX-1 in the mediation of cell proliferation.
Further mechanistic evidence was obtained from microarray analysis of genes involving in signaling pathways following PDX-1 knockdown in human PANC-1 cells, which have high endogenous expression of PDX-1. The computerized analysis of the microarray data also helped to identify additional potential molecular targets involved in the PDX-1 pathway, which provides potential insight into the molecular mechanism of PDX-1 in tumorigenesis. These data are consistent with our findings that PDX-1 regulates proliferation and invasion of PC cells and that suppression of PDX-1 expression via PDX-1 shRNA activates apoptotic pathways, as well as suppression of proliferation and invasion pathways28
. However, further studies are needed to determine precisely how PDX-1 regulates transformation.
In conclusion, the data in the present study demonstrate that PDX-1 overexpression resulted in: i) increased cell proliferation and invasion, as well as transformation of non malignant human cells; ii) promotion of tumor formation and growth of human cells implanted in SCID mice; and iii) disruption of the cell cycle in non malignant cells. These data, along with the demonstration that PDX-1 is over-expressed in more than 80 pancreatic cancer specimens, support the hypothesis that PDX-1 is an oncogene mediating tumorigenesis in pancreatic cancer.