There has been an absence of live human cell models of PDAC progression and consequently little information about proteins that could serve as released biomarkers and pathway indicators for early stages of the disease. As noted in the Introduction, when human PDAC or pancreatic cancer stem cells are grafted into immunodeficient mice, tumors rapidly arise that resemble the advanced PDAC stages from which the cells were derived and they do not undergo the slow growing phenotype of PDAC precursors. We hypothesized that, based on the ability of certain cancer cells to be reprogrammed to pluripotency by nuclear transfer and then to undergo early mammalian development, pluripotent stem cell lines from human pancreatic tumors might have the capacity to progress through early developmental stages of the cancer. This would provide an opportunity for discovering intrinsic processes and secreted protein biomarkers of live, early-stage human cells for a devastating cancer. Indeed we showed that a rare, single pancreatic cancer iPS-like line, 10-22 cells, can provide novel insights into human cancer progression.
How does the ectopic expression of Oct4, Sox2, Klf4, and c-Myc and a pluripotent-like state suppress the cancer phenotype? Apparently the pluripotency epigenetic environment can dominate over certain oncogenic states (Lonardo E, 2011). In nuclear transfer studies, only certain cancer cells are amenable to reprogramming (Blelloch et al., 2004
; Hochedlinger et al., 2004
; Li et al., 2003
) and, similarly, we only obtained one iPS-like line from pancreatic cancer harboring a KRAS
mutation, the predominant driver of PDAC. While the KRAS
mutation induces MAPK signaling, which can trigger mouse ES cells to differentiate (Kunath et al., 2007
), in human ES cells, MAPK signaling can promote self-renewal (Eiselleova et al., 2009
). Oncogenic RAS
induces cellular senescence by the accumulation of p53 or CDKN2A (Serrano et al., 1997
) and the expression of the four reprogramming factors also triggers senescence by inducing p53 and CDKN2A, thereby impairing reprogramming (Banito et al., 2009
). Only patient #10 in our study had a deletion in exon2 of CDKN2A, possibly explaining how the 10-22 cells could escape a senescent phenotype. Additional mutations could have arisen in the 10-22 cells that made the cells particularly amenable to iPS formation. Further work will determine what kind of mutations predict whether a cancer cell can be reprogrammed to pluripotency.
How does release from pluripotency allow the cancer genome to be expressed in a stage-specific fashion, as opposed to undergoing an immediate regression to the late stage phenotype? While the answers to these questions are not in hand, release from pluripotency is normally accompanied by the development of germ layer cells and then specialized tissues, which may continue to dominate, epigenetically, over the resident cancer genome (Blelloch et al., 2004
; Hochedlinger et al., 2004
; Li et al., 2003
). The 10-22 cells from PDAC generated diverse tissue types in teratomas as well as pancreatic ductal tissue that exhibited PanIN lesions and later progression. The apparent preference for pluripotent cells to regenerate the cancer type from which they were derived reflects the tendency of iPS cell lines in general to preferentially differentiate into their lineages of origin (Bar-Nur et al., 2011
; Kim et al., 2011
Several lines of evidence indicate that the 10-22 iPS-like line is derived from PDAC. First, the pathology of the original, recurrent tumor was that of PDAC and the CGH profile of the bulk population of cultured cells, which had a highly disrupted genome, was represented in the CGH profile of the 10-22 iPS-like line (, S3B
). While the tumor harbored pockets of more differentiated epithelial cells amidst a vast majority of undifferentiated cells, and therefore it is not certain which type of cell was immortalized in the 10-22 line, the 10-22 cells’ disrupted genome does reflect that of a typical epithelial cell in the recurrent tumor. Second, the PanIN-like structures from the 10-22 cells’ teratomas expressed SOX9 (Figure S5B-E
), which is required for early, KrasG12D
-dependent pancreatic precursor lesions in a mouse model (Kopp et al., 2012
), as well as PDX1, a definitive pancreatic cancer epithelial marker. Thus the ductal lesions from the 10-22 cells are of a pancreatic type. Third, teratomas at 9 months from 10-22 cells progressed to the histology and locally invasive characteristics of later stage PDAC (). Thus, the 10-22 line was not from an early stage cell that would solely undergo an early stage phenotype. Taken together, the evidence indicates that the 10-22 iPS-like line is from PDAC cells in the original tumor and that, upon re-differentiation in teratomas, it undergoes progression of the disease. This is unlike other human PDAC lines, which exhibit late stages of cancer (Lieber et al., 1975
; Yunis et al., 1977
Could the 10-22 iPS-like line be derived from cancer stem cells within the original tumor? Pluripotency genes such as NANOG
are expressed in sphere cultures of pancreatic cancer stem cells (CSCs), suggesting that such cells might be more susceptible to reprogramming (Lonardo et al., 2011
). However, CD133+CXCR4+ pancreatic CSCs are not enriched and the expression of pluripotent genes is not observed in the adherent culture conditions we used to derive the 10-22 cells (Hermann et al., 2007
). Also, the OCT4
pluripotency genes were highly methylated in the parental tumor #10 epithelium cultures, in contrast to the 10-22 cell line and the huES H1 control, and NANOG itself was not expressed in the primary tumor, although OCT4 was expressed sporadically (Fig. S2B, C
). Taken together, it seems unlikely that 10-22 cells were derived from pancreatic CSCs. In addition, pancreatic CSCs (Hermann et al., 2007
; Ishizawa et al., 2010
; Li et al., 2007
) rapidly generate aggressive tumors that represent the primary tumors; whereas the 10-22 cells generate slow growing PanINs (, S4
). Finally, tumors generated with pancreatic CSCs give rise to both cytokeratin negative and positive cells in the resultant tumors, in contrast to the homogenous K19 positive staining in PanIN-like ducts at 3 month teratomas (, S2A
). Thus, 10-22 cells appear not to exhibit properties of pancreatic cancer stem cells.
The proteins released or secreted from the PanIN-like teratomas fell into 3 major networks, including inter-connected networks for TGFβ and integrin signaling that suppress PDAC progression (Hezel et al., 2012
). We also report here for the first time the activation of an HNF4α network distinctive for the late PanIN stages. HNF4α is not or barely expressed in normal pancreatic ductal cells, poorly expressed in the PanIN1 stage, but is activated in PanIN2 and PanIN3 stages, invasive stages, and in early well-differentiated human pancreatic cancer. HNF4α levels then decrease markedly in advanced or undifferentiated PDAC. We found that these expression states also occur in a mouse model of PDAC progression. Dynamics in HNF4α expression affect the oncogenic transformation of liver cells (Hatziapostolou et al., 2011
). It remains to be determined whether the expression of HNF4α and its target genes is a cause or consequence of pancreatic cancer progression. Yet considering that pancreatic cancer is typically discovered in advanced or metastatic stages, activation of HNF4α and the release or secretion of proteins from the factor's target genes specifically in the late PanIN stages should provide useful diagnostics.
Of 107 proteins reproducibly released or secreted from PanIN-like cells derived from the 10-22 line, a total of 68 proteins overlap with genes, proteins, and networks expressed in human PanIN and PDAC, further validating the origin of the cells. In addition, we show that such proteins are released from the cells and stable, thus serving as potential biomarkers of early PDAC. A subset of the secreted proteins could be from locally activated stromal cells in the explants. We suggest that the combined detection of released proteins that are the products of the HNF4α, TGFβ, and integrin networks within PanIN and invasive PDAC cells may provide the best means for noninvasively detecting the progression of pancreatic cancer in humans.
Based on our extensive efforts with diverse initial PDAC samples (see Tables S1, S2), iPS cells arising from epithelial cells of solid tumors appear to be rare events, possibly involving secondary changes that allow iPS formation. It is therefore unclear whether the iPS approach will work with other solid tumor samples. Despite these caveats, the 10-22 cells behaved in a highly consistent fashion and this study revealed novel information about human PDAC. It is hoped that a better understanding of how to create iPS cells from human epithelial cancers will provide opportunities in the future with other types of solid tumors.