Regulation of G1 cell-cycle progression by oncogenes and tumor suppressor genes

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Regulation of G1 cell-cycle progression by oncogenes and tumor suppressor genes Alan Ho and Steven F Dowdy* Progression of resting quiescent G0 cells into early G1 and transition across the restriction point are highly regulated processes. Mutation of proto-oncogenes and tumor suppressor genes regulating these transitions are targeted during oncogenesis. Recent work has underscored the importance of the G0 to early G1 transition and metabolism to neoplastic cells. Addresses Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, UCSD, 9500 Gilman Drive, La Jolla, California 92093-0686, USA *e-mail: [email protected] Correspondence: Steven F Dowdy Current Opinion in Genetics & Development 2002, 12:47–52 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations cdk cyclin-dependent kinase HDAC histone deacetylase MEFs mouse embryonic fibroblasts Rb Retinoblastoma

Introduction Cell proliferation is an ordered, tightly regulated process involving multiple checkpoints that assess extracellular growth signals, cell size, and DNA integrity. The somatic cell cycle is divided into an interphase, designated for cellular growth and DNA synthesis, and a mitotic phase, in which a single cell divides into two daughter cells. Interphase is further subdivided into two gap phases (G1 and G2) separated by a phase of DNA synthesis (S phase). However, the vast majority of cells in the human body exist in a non-dividing, terminally differentiated state. In contrast, stem cells exist in a resting G0 quiescent state. Upon appropriate external stimulation, G0 cells can enter the cell cycle into early G1, likewise cycling cells present in early G1 can exit into G0 when deprived of external growth stimulus [1]. Studies of tumor cell growth kinetics have revealed that at least three parameters are critical to tumor growth in vivo: first, the duration of a single cell cycle; second, the proportion of cells actively proliferating (known as the ‘growth fraction’); and third, the rate of cell loss as a result of death or apoptosis [2]. Surprisingly, the length of tumor cell cycles measured in vivo and in vitro are not significantly shorter than those of normal cells, suggesting that neoplastic cells are acquiring genetic alterations that either enhance the growth fraction and/or minimize cell loss, rather than simply decreasing the cell-cycle duration. Consequently, processes that govern cell-cycle entrance/exit and metabolism are

perhaps more important to tumor growth than those events that regulate the rate of individual cell division. These ideas help to understand the consequences associated with alterations of the cell-cycle machinery during oncogenesis. The point between the early G1 and late G1 phase passage that represents an irreversible commitment to undergo one cell division is termed the ‘restriction point’ [1]. Importantly, the restriction point divides the cell cycle into a growth factor dependent early G1 phase and growth factor independent phases from late G1 through mitosis [1]. Growth factor signaling determines whether early G1 phase cells transit the restriction point to undergo eventual cellular division or, because of insufficient signaling strength, exit the cell cycle, enter into G0. Thus, although overcoming growth factor signaling dependency is a major hurdle in the development of neoplastic disease, this requirement actually serves at least three significant purposes: first, avoidance of G0 exit; second, sustained metabolism; and third, transition across the restriction point into late G1 (Figure 1). Surprisingly, the role and significance of growth-factor signaling required to avoid cell cycle exit and sustain metabolism during oncogenesis has been greatly overlooked in tumor biology studies.

The retinoblastoma protein family Retinoblastoma (Rb) is a rare childhood malignancy that serves as the classic model for the loss of tumor suppressor function, namely pRb (Rb protein), in oncogenesis [3]. pRb is a key negative regulator at the restriction point. Surprisingly, with the exception of three relatively rare malignancies (retinoblastoma, osteosarcoma, and small cell lung carcinoma) [3], the overall rate of Rb mutation in the vast majority of human cancers is either extremely low or non-existent, suggesting that direct inactivation of pRb is not an easily sustainable genetic ablation. Consistent with these observations, homozygous germline deletions of murine Rb results in an early embryonic lethality [4–6]. Therefore, overriding the restriction point simply by removal of the key negative regulator does not promote oncogenesis, but rather results in unscheduled apoptosis during differentiation [4–8]. One important pRb target for regulating early G1 cell cycle progression is the E2F family of transcription factors [9]. E2Fs regulate the expression of a host of genes mediating both restriction point traversal (cyclin E), and S phase progression (dihydrofolate reductase and thymidylate synthase) [9]. E2Fs are regulated during G0 and S phase by two pRb family members — p130 and p107, respectively — that are rarely, if ever, targeted during oncogenesis [9].

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Oncogenes and cell proliferation

Figure 1

Growth factor dependent phase

G0

Growth factor independent phases

Late G1

Early G1

S phase

Restriction point

G0 resting cell

Early G1 cell

Late G1 cell

S-phase cell

• Small cytosol

• Increasing cytosol

• Increasing cytosol

• Increasing cytosol

• Low metabolism

• High metabolism

• High metabolism

• High metabolism

• Dividing mitochondria

• Dividing mitochondria

• Dividing mitochondria

• Commitment to divide

• Replicating genomic DNA

• DNA synthesis genes expressed

Resting G0 quiescent cells have a low metabolism and a small cytosolic volume compared to their dividing counterparts. Upon growth factor stimulation, the resting cells first traverse across the reversible G0 to early G1 transition where metabolism is dramatically increased and cell volume increases. Continued metabolic output and cell cycle progression during early G1 is growth factor dependent. After achieving an appropriate cell size, early G1 cells irreversibly cross the restriction point into the growth factor independent late G1 phase and are committed to undergoing DNA replication (S phase) followed by mitosis.

Current Opinion in Genetics & Development

Rb knockout mouse embryonic fibroblasts (MEFs) possess deregulated and elevated expression of a number of E2F targets [10–12]. Surprisingly, Rb–/– MEFs demonstrate only a slight decrease of G1 cell number among an asynchronously dividing population in culture, suggesting the presence of a pRb-independent event(s) that must be satisfied prior to restriction point transition [10,12]. In contrast, serum-deprivation and re-stimulation (addition of serum) of Rb–/– MEFs results in a significantly increased rate of cell-cycle entry compared to wild-type MEFs [10–12]. Taken together, these observations suggest that serum-deprived Rb–/– MEFs either fail to exit the cell cycle into G0 or they more readily traverse from G0 into early G1 phase leading to the conclusion that the critical role of pRb in regulating cell-cycle progression is the maintenance of early G1 and not G0 maintenance. By preserving a proper early G1, pRb would be allowing the cell to complete at least two critical tasks before committing to a complete round of division: first, integration of pro- and anti-growth signals and second completion of cellular metabolism and growth necessary to sustain cell size and, hence, continued proliferation. Interestingly, G1 Rb–/– MEFs were also significantly smaller in size relative to wildtype, suggesting that a shortened G1 resulted in a failure to accumulate the necessary cellular mass for proper division [10]. Eventually, signals from these processes of cell growth and metabolism must also feedback to execute pRb inactivation to ensure a timely and regulated progression through each cell cycle.

Cyclin–Cdk complexes during G1 Although pRb is a negative regulator of early G1 cell-cycle progression, it is paradoxically regulated by the positive regulators of cell-cycle progression, namely cyclin–cdk complexes. Cyclin–cdk complexes are an evolutionarily conserved family of proline-dependent serine/threonine kinases [13]. During G1, two predominant cyclin–cdk complexes are active, namely cyclin D/cdk4/6, comprising three D-type cyclins (D1–3) and two different cdk subunits (cdk4 and cdk6), and cyclin E/cdk2 [13]. Cyclin D/cdk4/6 complexes are constituitively active throughout early and late G1 of dividing cells [14••]. In contrast, cyclin E–cdk2 complexes are activated concomitant with transition across the late G1 restriction point and the appearance of hyperphosphorylated pRb [14••,15–17]. Consequently, these observations suggest that cyclin D and cyclin E complexes perform non-overlapping and functionally independent functions during early G1. Importantly, cyclins D1, D2, Cdk4 and Cdk6 are bona fide human protooncogenes selected for overexpression during oncogenesis and hence, tolerable to the neoplastic cell [18]. In contrast, cyclin E and cdk2 are rarely, if ever, targeted. On the basis of early studies, a model of G1 cell cycle progression has emerged whereby cyclins D and E were thought to mediate functionally equivalent sequential, inactivating phosphorylation events on pRb [19,20]. However, this interpretation directly contradicts the fundamental physiological finding that cyclin D/cdk4/6

Regulation of G1 cell-cycle progression Ho and Dowdy

complexes are constituitively active throughout G1 phase in both cycling normal and tumor cells, whereas pRb remains in its active, hypophosphorylated form bound to E2Fs during early G1 [14••]. In contrast, cyclin E–cdk2 activity demonstrates regulated cell-cycle periodicity that occurs concomitant with pRb hyperphosphorylation and inactivation at the restriction point [14••,20]. Moreover, loss of cyclin E activity results in active, hypophosphorylated pRb bound to E2Fs and failure to cross into late G1 [14••,20,21]. Recently, a new oncogenic role for cyclin D/cdk4/6 complexes has been proposed whereby cyclin D activity partially inactivates pRb function by disrupting complexes of pRb and histone deacetylase (HDAC) leading to de-repression of the cyclin E gene and consequential activation of cyclin E/cdk2 [19,22–24]. However, and importantly, this notion, based on transfection experiments [19], directly contradicts the three original papers describing endogenous pRb–HDAC associations in transformed and tumorigenic cells containing physiologically deregulated cyclin D/cdk4/6 activity due to p16 loss (a negative regulator of cdk4/6) [22–24]. Thus, the presence of endogenous pRb–HDAC complexes in p16 null tumor cells is not consistent with a putative cyclin D/cdk4/6 role in regulating pRb–HDAC interactions. The confusion surrounding cyclin D/cdk4/6 regulation of pRb partially stems from the lack of a consensus for what active pRb actually is. Several studies have shown that hypophosphorylated pRb in G1 is the active form that binds E2F transcription factors and represses expression of E2F target genes [14••,25,26]. In contrast, unphosphorylated pRb in G0 does not associate with E2Fs and is considered inactive [14••,25–27]. Recently, Ezhevsky et al. [14••] have shown that cyclin D activity hypophosphorylates and activates pRb as a transcriptional repressor in early G1, whereas cyclin E activity inactivates pRb by hyperphosphorylation at the restriction point. Moreover, genetic studies in Drosophila point to a role for cyclin D/cdk4 in promoting cell growth and metabolism during G1 and not a role in progression across the restriction point into S phase [28••,29]. Thus, coupled with the original data demonstrating induction of cyclin D activity upon growth-factor stimulation [30,31], it now becomes clear that cyclin D activity both hypophosphorylates pRb in promoting the G0 to early G1 transition and increases metabolism, necessary to sustain uncontrolled divisions in neoplastic cells.

Cyclin–Cdk inhibitors during G0 and G1 Whereas cyclin–cdk complexes are key regulators of G1 cell cycle progression, evolution has added yet another layer of G1 cell cycle regulation in the form of cyclin–cdk inhibitory proteins. At present, two classes of G1 cyclin–cdk inhibitors, the INK4 and Cip/Kip families, operate in distinct fashions to regulate G1 cyclin–cdk complexes. The INK4 family, comprising p15, p16, p18 and p19 proteins, specifically bind monomeric cdk4/6 preventing cyclin D activation, whereas Cip/Kip family members, p21, p27 and p57 proteins, bind and inactivate heterodimeric

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cyclin–cdk2 complexes [32]. Surprisingly, genetic mutations have only been detected in p16 — loss of the p15 gene only occurs concomitant with loss of the proximal p16 gene — whereas epigenetic alterations have been reported for p27, again suggesting that loss of function of these two genes results in a selective and tolerable advantage [18,32]. Although the mechanism of p16 cell cycle arrest has been somewhat obfuscated by work demonstrating a requirement for cyclin E inhibition by the Cip/Kip family [33], the undisputed hypothesis is that accumulation of p16 protein in a physiological setting sequesters cdk4/6, driving cells out of the cell cycle into G0 and/or preventing cycling cells from avoiding cell-cycle exit [32]. Thus, the frequent deletion of INK4 proteins again suggest that oncogenesis selects for genetic alterations that bypass mechanisms meant to force cells to exit the cell cycle [18]. Importantly, these human tumor observations are correspondingly confirmed in p16 knockout mice that are viable and suffer only a subtle tumorpredisposition phenotype [34•,35]. Although p27 is rarely, if ever, genetically targeted in human malignancies, low expression of p27 in tumor samples has been correlated to poor prognoses in breast carcinoma, colon carcinoma, and other malignancies [32]. Moreover, cytosolic localization of p27 has been correlated with increased tumor aggressiveness [36]. However, as p27 is signaled for increased degradation [37,38] and cytosolic localization in dividing cells, these correlations may have little to do with direct epigenetic selection for p27 loss of function during oncogenesis and may be consequence of increased degradation as a result of a higher fraction of dividing cells in aggressive tumors. Several cell culture studies have shown that p27 is critical to the maintenance of G0 [39–42], and that it may also play a role in cyclin E activation at the restriction point [38,41]. Recent work has shown, however, that p27-deficient MEFs show little, if any, alteration in the timing of restriction point traversal compared to wild-type MEFs. In addition, p27/p21 doubly deficient MEFs also do not alter the timing of pRb hyperphosphorylation. Moreover, p27 degradation is dependent on phosphorylation by cyclin–Cdk2 complexes, placing p27, in part, as a downstream substrate of cyclin–Cdk2. It has been demonstrated recently that Skp2 and Cks1 are critical components of the ubiquitin-dependent p27 degradation machinery, highlighting the importance of maintaining proper p27 degradation to regulate protein turnover [43–47]. Nevertheless, these studies fail to elucidate either of these components as the rate-limiting regulator of p27 levels and function, as both presumably function downstream of cdk2 phosphorylation. Indeed, inhibition of cdk2 activity in synchronized cells results in p27 stabilization (A Ho, SF Dowdy, unpublished observation), placing the kinase upstream all p27-degradation machinery. Alexander and Hinds [48•] have demonstrated recently that p27 is a downstream effector of pRb-mediated senescence, further suggesting that p27 plays a critical role in mediating cellcycle exit, not restriction point regulation in cycling cells.

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Oncogenes and cell proliferation

Figure 2

Growth factors

Metabolism

?

p16

Cyclin D–Cdk4/6

Go

p27

Cyclin E–Cdk2

Late G1

Early G1 Restriction point

pRB

p130 E2F

Regulation of G1 cell cycle progression. Tumor cells preferentially select for alterations in p16, cyclin D (D1, D2), Cdk4, and Cdk6 (in red) which are involved in regulating the G0 to early G1 transition, whereas genes negatively regulating restriction point traversal, such as p27 (in blue) and pRb, as well as positive regulators at the restriction point, including cyclin E and Cdk2, are rarely, if ever, targeted during oncogenesis. p130:E2F:HDAC complexes either directly or indirectly transcriptionally repress genes involved in cell cycle progression and metabolism, whereas pRB:E2F:HDAC complexes repress only cell cycle genes.

HDAC

E2F

HDAC

Late G1 genes Metabolic genes Current Opinion in Genetics & Development

The potentially dual roles of p27 in both maintaining G0 and regulating restriction point transition leaves open speculation as to how decreased expression of p27 leads to increased tumor aggressiveness. p27 knockout mice have a relatively subtle phenotype of gigantism (~30% increased weight) and tumor predisposition [49–51]. Thus, although mouse models indicate that p27 has the potential to act as a tumor suppressor, such direct genetic evidence has yet to be observed in human tumor studies. Thus, consistent with other cell-cycle genes targeted during oncogenic selection discussed above, alteration of p16 and p27 function appear to engender a tolerable phenotype on the neoplastic cell.

Conclusions An increasingly precise model for the regulation of G1 cell cycle progression in normal and neoplastic cells is emerging from recent genetic and biochemical studies that incorporate the observed physiological activities of cell-cycle regulatory proteins in normal and neoplastic cells (Figure 2). These studies draw our attention to the significance of disrupting regulation of the G0 to early G1 progression in neoplasia, rather than those events involved directly in restriction point traversal. Importantly, cell-cycle components selectively targeted during oncogenesis appear to regulate the growth factor dependent G0 to early G1 transition and thereby avoid cell-cycle exit as well as increased metabolism. Conversely, alterations in components regulating restriction point traversal and S-phase entry do not appear to contribute to tumorigenesis but instead appear to influence the level of tumor cell proliferation.

Similar to observations in primary human tumors, mice genetically modified for gene products involved in regulating the G0 to early G1 transition (viz. p16, p27, cyclin D, and cdk4) produce viable mice with relatively subtle tumor-predisposition phenotypes. In stark contrast, modification of genes that increase the rate of cell-cycle progression at the restriction point (viz. pRb, cyclin E, and cdk2) result in embryonic lethality. Hence, genetic alterations in cancer must balance the consequential strength of the mutation to provide a growth advantage to the tumor cell while simultaneously avoiding apoptotic induction. Importantly, this model highlights the need to preserve pRb function during the initial stages of oncogenesis as opposed to inactivating pRb. In addition, pRb maintenance of a proper, early G1 is potentially important for maintaining the cell growth and metabolism necessary for division. Furthermore, for oncogenesis, the role that constitutive cyclin D kinase activity plays in driving cell growth and metabolism in early G1 may very well be of equal importance to its role in promoting G0 to early G1 progression. Although much must still be learned, significant progress has been made recently in understanding the involvement of cell-cycle alterations during oncogenesis. Future studies will undoubtedly result in new insights that will help fine tune existing models of cell-cycle progression.

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