Dormant tumor-suppressor pathways in tumors.

Abstract

It was initially thought that activation of a single oncogene such as Ras would result in tumorigenesis; however, this conclusion was based on studies in which immortalized cells were used as the target cells in transformation assays (1). Thus it was significant when in 1983 Newbold and Overell reported that an activated Ras gene failed to transform normal fibroblasts unless the fibroblasts were first “immortalized by carcinogens” (2). On the basis of these and similar findings, Newbold and Overell and several other groups insightfully hypothesized that Ras gene activation was only one of a number of mutations necessary for the “progression to malignancy” (2, 3). This was borne out when it was found that two different oncogenes that failed to transform normal fibroblasts on their own could cooperate to transform the fibroblasts when expressed together (4, 5). These findings provided the first in vitro evidence that tumorigenesis is a multistep process requiring the activation of several oncogenes. We now also understand that the process of tumorigenesis not only involves mutations that activate oncogenes; it also requires several mutations that disrupt the activities of proteins that function to suppress tumorigenesis (6). Among these are the cyclin-dependent kinase (cdk) inhibitor p16 INK4a and the paradigm tumor-suppressor protein p53. Indeed, the p16 INK4a and p53 genes are among the most frequently mutated genes in human cancers. When activated by inappropriate proliferative signals or damage to the cell’s genome, the proteins expressed by these genes halt cell-cycle progression. Thus, these proteins prevent the proliferation of cells that had developed the potential to undergo transformation. p16 INK4a arrests cells by inhibiting cyclin D/cdk activity. Cyclin D/cdk activity is necessary to maintain the tumor-suppressor retinoblastoma protein (pRb) in its inactive form, so inhibition of cyclin D/cdk results in arrest mediated by active pRb. The activation of p53 results in increased expression of p21 WAF1 , a cdk inhibitor that induces cell-cycle arrest. Hence, if p16 INK4a or p53 are inactivated, important constraints on inappropriate proliferation are lost. In the context of our current understanding of the function of tumor-suppressor proteins, previous findings regarding a seemingly paradoxical effect of Ras can be rationalized. Expression of oncogenic forms of Ras will transform most immortalized cell lines; however, in some cells, rather than induce transformation, oncogenic Ras will arrest proliferation (7, 8). This occurs when it is expressed in either normal human or normal rodent fibroblasts, as well as in some immortalized cell lines. Arrest of cell proliferation is mediated by tumor-suppressor pathways, since p16 INK4a and p53 protein levels increase when oncogenic Ras is expressed in fibroblasts, and the fibroblasts undergo transformation, instead of arrest, if these proteins are inactivated (7). Thus when the cell senses an inappropriate proliferative signal, as it would if its endogenous Ras gene were mutated to an oncogenic form, it responds by activating its tumor-suppressor activity. The mechanism by which tumor-suppressor pathways are activated by oncogenic Ras is not known. Ras binds to and activates several effector proteins. The interaction of Ras with one of these proteins, Raf, appears to be sufficient for both Ras-induced transformation and cell-cycle arrest. The interaction of Ras with Raf activates MAP/ ERK kinase (MEK), which in turn activates mitogen-activated protein (MAP) kinases. In most immortalized cells, constitutive activation of this pathway results in the upregulation of cyclin D/cdk activity and the downregulation of cellular levels of p27 KIP1 , another cdk inhibitor that can mediate cell-cycle arrest. These changes ultimately result in cellular proliferation (9). When Ras is activated in normal fibroblasts or in immortalized cells in which growth is arrested in response to Ras, the same pathway is activated through the MAP kinase. However, in these cells this signaling results in growth arrest mediated by either p16 INK4a , p21 WAF1 (as a result of p53 activation), or p27 KIP1 , depending upon the cell type (10). The mechanism by which this alternative, growth-inhibitory pathway is activated has not been delineated. In this issue of the Journal , Ravi and colleagues address the finding that even though Ras mutation is common in human cancers, it is mutated in less than one percent of small-cell lung cancers (SCLC) (11). They previously have reported that constitutive activation of the Ras–MAP kinase pathway results in the growth arrest of two pRb-negative SCLC cell lines (12). This arrest correlates with an increase of p27 KIP1 , a cdk inhibitor that can arrest cellular proliferation in a pRb-independent manner. In their current work, Ravi and coworkers demonstrate that constitutive activation of the Ras–MAP kinase pathway will also cause a growth arrest of a pRb-positive SCLC cell line. In contrast to the Rb-negative SCLC cell lines they studied ( Received in original form March 18, 1999 )