Mutational activation of Ras is one of the most common oncogenic events detected in human cancers. Although accumulating evidence suggests that Ras activation is an important event in the initiation of tumors of the lung, pancreas, and colon (3
) and many of the downstream effectors of Ras have now been identified, its precise role in cancer initiation and progression is still unclear. Moreover, the observation that the vast majority of oncogenic Ras alleles arise in the Kras isoform, as opposed to the very closely related Hras and Nras isoforms, has yet to be explained mechanistically. Here, we provide evidence from cell culture studies that mutationally activated Kras, but not Hras or Nras, can promote the expansion of an endodermal stem/progenitor cell population and prevent differentiation. These observations can potentially explain the relatively high frequency of Kras mutations in human cancer, and they reveal a possible role for Kras activation as an initiating event in tumorigenesis. Moreover, our findings are consistent with recent observations in mouse studies in which it was demonstrated that mutational activation of Kras, but not Nras, promotes expansion of the stem cell compartment of the mouse colon (K. Haigis and T. Jacks, MIT, personal communication), thereby supporting the physiological relevance of these cell culture studies.
The F9 cell culture model faithfully recapitulates the differentiation of a stem/progenitor cell to early endoderm. The fact that RA treatment can specifically drive this process in culture and that RA signaling is required for the formation of early endoderm in vivo also supports the physiological relevance of the model. Previous studies have demonstrated that mutationally activated Hras is sufficient to promote the differentiation of F9 cells to primitive endoderm in the absence of RA in transfection studies (86
). Our findings are consistent with those reports, and this observation can potentially explain why activating Hras
mutations are not seen in tumors of endodermal origin, such as pancreatic, lung, and colorectal cancers. Notably, activated Hras has also been reported to promote the differentiation of several other lineages, including adipocytes (8
) and neurons (5
). However, a role for activated Kras in these settings has been largely unexplored. Our observation that Hras, Nras, and Kras exert very different functions in the context of stem/progenitor cell differentiation suggests that it may be informative to compare the activities of the various Ras isoforms more broadly in the context of differentiation.
The observed differences in differentiation phenotypes associated with distinct Ras isoforms may be related to previous reports implicating tissue-dependent contexts in susceptibility to the transforming activity of particular oncogenes. Thus, lineage-specific factors associated with differentiation programs may contribute to the susceptibility of different tissue types to tumorigenic conversion by various oncogenes and to the resultant transformed phenotype (39
). For example, such differences may underlie the observations that activated Nras is associated with myeloid malignancies (66
), germ cell tumors (34
), congenital melanocytic nevi, and cutaneous melanomas derived from neural crest, but not mucosal melanomas, which are not derived from neural crest (7
). In addition, Hras activation is associated with tumorigenesis of the bladder (45
) and salivary gland (97
), tissues that arise from an ontogenetic transitional zone, a region where endoderm and ectoderm meet. Notably, the expression of activated Hras, Nras, or Kras in PCC4 cells, which can give rise to mesenchymal (but not endodermal) cells in culture, does not result in an observable phenotype. Together, these findings suggest that the distinct tumor types associated with mutational activation of the various Ras isoforms reflect the unique ability of each of the Ras proteins to affect the differentiation program of progenitor or stem cells that differentiate along distinct lineages.
The proposed model for activated Kras function in stem cells or partially committed progenitors, as an initiating step in human oncogenesis, would seemingly imply that “mature” tumors might continue to express stem cell markers. However, while Oct3/Oct4 expression in human tumors has previously been reported (60
), the expression is typically seen only in a very small fraction of tumor cells (53
), possibly cancer stem cells. This can be explained by either of two mechanisms. First, it is possible that a small fraction of cells within a tumor maintain stem cell characteristics and these cells are needed to continuously “replenish” the bulk of the tumor with progeny cells that exhibit a more differentiated phenotype. However, it is also possible that an activated Kras allele is needed to expand the stem cell population early in tumorigenesis, and subsequently, mutant Kras is selected for its ability to contribute to other aspects of tumor progression, while its role in stem cell maintenance is diminished. Indeed, activated Kras has been shown to promote proliferation, survival, and invasive properties in a variety of non-stem cell contexts (29
It is worth noting that the observed effects of the various mutationally activated Ras proteins on stem cell differentiation do not necessarily reflect a normal requirement for Ras proteins in stem cell maintenance or differentiation. However, the ability of KrasV12 to expand stem cells could be related to the reported studies describing a unique requirement for endogenous Kras, but not Hras or Nras, in mouse embryonic development (46
). Despite the ubiquitous expression of Ras isoforms in various tissues, the lack of correlation between expression and malignancy, and the fact that the various isoforms interact with the same constellation of effectors (2
), distinct cellular consequences of activating the various Ras family members have been reported (67
). This may reflect differential activation of effectors and/or differential signal intensity/duration (24
). Indeed, Ras effectors themselves can exhibit seemingly opposing effects, depending on the cellular context (22
). Such opposing activities are consistent with our findings that some Ras effectors can mediate distinct (and seemingly opposing) phenotypic consequences in the F9 model.
Such differences in isoform-dependent Ras signaling appear to largely involve the differential subcellular localization/processing of Ras and its effectors (15
). For example, a recent report indicates that oncogenic Hras-induced senescence is mediated by the endoplasmic-reticulum-associated, unfolded protein response (26
). Our studies with chimeric Ras isoforms and the Raf effector containing carboxy-terminal motifs that cause distinct subcellular localization of signaling complexes support a critical role for compartmentalized signaling in the differential biological activity of the Hras and Kras isoforms (Fig. ). However, it is also possible that the carboxyl termini of the various Ras proteins also contribute to their distinct biological effects through unique interactions with cellular proteins that have yet to be identified. Such a possibility is supported by our findings that the Kras chimera containing the extreme C terminus of Hras can maintain stem cell characteristics in RA-treated F9 cells and that the activated Kras-4A splice isoform induces apoptosis (Fig. ).
FIG. 10. Structure-function analysis of various Ras mutants and chimeric proteins in F9 cell differentiation, stem cell renewal, proliferation, and apoptosis. Indicated to the left of each wild-type or mutant protein structure is the wild-type or mutated Ras or (more ...)
The Raf, PI-3 kinase, and RalGDS proteins are important Ras effectors that contribute to Ras-mediated proliferation and survival or differentiation (50
). We observed that inhibiting PI-3 kinase can prevent either differentiation or self-renewal, depending upon the Ras isoform activated. Interestingly, it has also been reported that Ras isoform-dependent transformation potential correlates with PI-3 kinase activation (52
). Similarly, we observed that PI-3 kinase activity mediates expression of the POU transcription factor Oct3/Oct4, a homeobox gene, which has direct consequences for self-renewal or differentiation of F9 cells. Moreover, our analysis of Kras effector domain mutants further supports a role for the Raf and PI-3 kinase pathways in endodermal progenitor expansion and reveals an additional important role for RalGDS activation, which has recently emerged as an important Ras effector in the context of human tumorigenesis (37
). Interestingly, specific activation of RalGDS has been shown to prevent Hras-mediated differentiation of PC12 and myeloid cells (36
Our findings with the Raf-KTail chimera and the pharmacologic MEK inhibitor revealed both MEK-independent and MEK-dependent functions of activated Kras in stem/progenitor cells. Interestingly, these findings are consistent with recent studies indicating that KrasV12-induced expansion of mouse colonic epithelial stem cells in vivo appears to be MEK dependent, while maintenance of the undifferentiated state of these cells is MEK independent (K. Haigis and T. Jacks, MIT, personal communication). Taken together, our signaling studies reveal complex and context-dependent roles for three key Ras effectors in the various aspects of stem/progenitor cell maintenance, proliferation, and differentiation (Fig. ). However, many additional Ras effectors have been identified, and it certainly remains possible that some of those additionally contribute to the distinct functions of activated Hras and Kras in this setting. In summary, the identification of a unique role for Kras in promoting stem/progenitor cell expansion and an inhibition of differentiation to primitive endoderm provides a potential explanation for the high frequency of Kras mutations in tumors of endodermal origin. Finally, the observed unique ability of Kras to expand a stem/progenitor cell population indicates a potentially important role for Kras activation in the initiation of tumorigenesis.