Transforming growth factor-β and breast cancer: Cell cycle arrest by transforming growth factor-β and its disruption in cancer
© Current Science Ltd 2000
Received: 7 February 2000
Accepted: 7 February 2000
Published: 1 April 2000
Altered responsiveness to extracellular signals and cell cycle dysregulation are hallmarks of cancer. The cell cycle is governed by cyclin-dependent kinases (cdks) that integrate mitogenic and growth inhibitory signals. Transforming growth factor (TGF)-β mediates G1 cell cycle arrest by inducing or activating cdk inhibitors, and by inhibiting factors required for cdk activation. Mechanisms that lead to cell cycle arrest by TGF-β are reviewed. Loss of growth inhibition by TGF-β occurs early in breast cell transformation, and may contribute to breast cancer progression. Dysregulation of cell cycle effectors at many different levels may contribute to loss of G1 arrest by TGF-β. Elucidation of these pathways in breast cancer may ultimately lead to novel and more effective treatments for this disease.
TGF-β is a potent inhibitor of mammary epithelial cell proliferation [1,2] and regulates mammary development in vivo [3,4,5]. Mammary-specific overexpression of TGF-β in transgenic mice can induce mammary hypoplasia and inhibit tumourigenesis [6,7,8]. Although normal human mammary epithelial cells (HMECs) are exquisitely sensitive to TGF-β , human breast cancer lines require 10-fold to 100-fold more TGF-β to produce an antimitogenic response, and some show complete loss of this effect .
Although loss of growth inhibition by TGF-β in human cancers can arise through loss of TGF-β production or through mutational inactivation of the TGF-β receptors and Smad signalling molecules [11,12], these defects are not observed in most arrest-resistant cancer lines. This observation, and the frequent appearance of resistance to more than one inhibitory cytokine in human tumours  emphasize the importance of the cell cycle effectors of growth arrest induced by TGF-β as targets for inactivation in cancer.
Cell cycle progression is governed by cdks, which are activated by cyclin binding [20,21] and inhibited by the cdk inhibitors [22,23]. The cdks integrate mitogenic and growth inhibitory signals and coordinate cell cycle transitions [24,25]. G1 to S phase progression is regulated by D-type cyclin-, E-type cyclin- and cyclin A-associated cdks (Fig. 1). B-type cyclin-associated kinases govern G2 and M phases. Both E-type and D-type cyclin-cdks contribute to phosphorylation of the retinoblastoma protein (pRb). Hypophosphorylated pRb binds members of the E2F and DP1 families of transcription factors, inhibiting these transcriptional activators and actively repressing certain genes. Phosphorylation of pRb in late G1 phase liberates free E2F/DP1, allowing activation of genes required for S phase (for review ).
Cyclin-dependent kinase regulation by phosphorylation
Cdk activation requires phosphorylation of a critical threonine (Thr160 in cdk2 and Thr187 in cdk4). There are two mammalian kinases with in vitro cdk activating kinase (CAK) activity: cyclin H/cdk7 and the protein encoded by the human homolog of the Saccharomyces cerevisiae CAK1, called Cak1p (for review ). The specific roles of these two kinases are somewhat controversial. CAK is active throughout the cell cycle [20,28], but its access to cyclin-bound cdks is inhibited by p27 . Cdks are also negatively regulated by phosphorylation of specific inhibitory sites . Cdc25 phosphatase family members must dephosphorylate these inhibitory sites for full cdk activation. Cdc25A acts on cyclin E-bound cdk2 and is required for G1 to S phase progression .
Cyclin-dependent kinase inhibitors
Two cdk inhibitor families regulate the cell cycle [22,23]. The inhibitor of cdk4 (INK4) family members (p15INK4B, p16INK4A, p18INK4C and p19INK4D) inhibit specifically cdk4 and cdk6. The p16 gene, or MTS1 (Multi-Tumor Suppressor 1), was discovered as a tumour suppressor that is deleted in many cancers . Loss of p15, located near p16 on chromosome 9p, may contribute to loss of G1 arrest by TGF-β (see below).
The kinase inhibitor protein (KIP) family presently consists of three members, p21WAF1/Cip1, p27Kip1 and p57Kip2. The KIPs bind and inhibit a broader spectrum of cdks than do the INK4s. p21 is low in serum-deprived quiescence, but p21 levels and p21 binding to D-type cyclin-cdk complexes increase in early G1 phase. In addition to regulating G1 phase progression, p21 acts to coordinate cell cycle responses to DNA damage . p27Kip1 was first identified as a heat stable protein whose binding to cyclin E-cdk2 complexes that was increased by TGF-β, lovostatin, or contact inhibition [32,33,34,35,36]. p27 is high in G0 and early G1 phase and decreases during G1 to S phase progression. p27 degradation by ubiquitin-dependent proteolysis  is activated by many different growth factors and may involve ras pathways [38,39,40,41,42]. Although cyclin E-cdk2 phosphorylates p27 on Thr187 leading to its degradation in late G1 phase [43,44], other kinases may also influence p27 function and/or degradation. The possibility that mitogenic signalling pathways that modulate p27 phosphorylation also oppose Smad activation by TGF-β is the subject of intensive investigation.
Although p21 and p27 inhibit cyclin E-cdk2, they also function in the assembly and activation of cyclin D-cdk4 and cyclin D-cdk6 complexes. KIP-mediated assembly of D-type cyclin-cdks in early G1 phase may facilitate activation of E-type cyclin-cdks through sequestration of KIPs away from cyclin E complexes [45,46].
Mechanisms of cell cycle arrest by TGF-beta
TGF-β inhibits phosphorylation of the retinoblastoma protein
Cells are sensitive to TGF-β during a discrete period in early G1 phase, until they reach a 'restriction point' 6-10 h after G0 release [47,48]. When TGF-β is added after this critical time point, cells complete the cell cycle but arrest during the subsequent G1 phase. Laiho et al  observed that TGF-β inhibits pRb phosphorylation when it is added in early G1 phase. This key observation suggested that TGF-β was acting before the G1 to S phase transition to inhibit a pRb kinase, and led to the investigation of TGF-β effects on cell cycle regulators. These studies have shown that TGF-β prevents or inhibits G1 cyclin-cdk activation through multiple mechanisms, leading to pRb dephosphorylation (Fig. 2). E2F activity is also impaired by TGF-β through a decline in E2F mRNA levels . The observation that E2F overexpression can prevent TGF-β-mediated arrest  emphasizes the importance of the effects of TGF-β on pRb and E2F.
TGF-β downregulates c-myc
In many cell types, TGF-β causes a rapid inhibition of c-myc transcription [2,16,50]. Transcriptional regulation by the c-Myc protein is required for G1 to S phase progression. Downregulation of c-myc by TGF-β is believed to be important for arrest, because c-myc overexpression causes TGF-β resistance [2,51]. Repression of the c-myc gene by TGF-β may directly or indirectly contribute to the loss of G1 cyclins [52,53], to downregulation of Cdc25A  and to the induction of the cdk inhibitor p15  (see below).
Effects on G1/S cyclins
TGF-β causes loss of G1 cyclins in a cell-type-dependent manner. Cyclin A expression is downregulated by TGF-β in most cell types [32,56] and a TGF-β-regulated region of the cyclin A promoter has been identified . Effects of TGF-β on cyclin E differ among different cell lines. For example, in HaCat keratinocytes TGF-β decreases both mRNA and protein levels of cyclin A and cyclin E, whereas in HMECs cyclin E mRNA is reduced but protein levels are not [32,56]. Although cyclin D1 levels are decreased by TGF-β in some cell types, this usually occurs late as a consequence of arrest [58,59].
Cooperation between p15 and p27
In epithelial cells, including HMECs, the INK4 and the KIP proteins collaborate to inhibit D-type cyclin-cdks and E-type cyclin-cdks to bring about G1 arrest by TGF-β [60,61]. p15 INK4B was first cloned as a gene upregulated by TGF-β  and its induction involves an Sp1 site in the promoter . TGF-β induces p15 INK4B and stabilizes the p15 protein, leading to p15 binding and inhibition of cdk4 and cdk6. Cyclin D1 and KIP molecules dissociate from cdk4 and cdk6, and p27 accumulates in cyclin E-cdk2 complexes, inhibiting the latter [60,61]. A late downregulation of cyclin D1 and cdk4 follows G1 arrest. TGF-β appears to actively regulate p27's affinity for its targets, independent of p15 function, favouring p27 accumulation in cyclin E complexes .
Upregulation of p21 expression
Effects on cdk2 phosphorylation
TGF-β also regulates cdk2 phosphorylation. In Mv1Lu cells, TGF-β inhibits cdk2 in part by inhibiting phosphorylation on Thr160 [32,34]. p27 can inhibit CAK access to cyclin-bound cdks in vitro . Thus, TGF-β may prevent CAK action by increasing the binding of p27 to cyclin E-cdk2. In HepG2 cells, however, TGF-β inhibits the enzymatic activity of Cak1p , indicating an alternative mechanism for the inhibition by TGF-β of Thr160 phosphorylation of cdk2.
Dephosphorylation of inhibitory sites on cyclin E-bound cdk2 is required for G1 progression and is required for G1 to S phase progression . In a human breast epithelial line, TGF-β reduced Cdc25A expression in association with an increase in inhibitory cdk phosphorylation . The effect on Cdc25A expression may be secondary to the repression by TGF-β of c-myc, because in some cell types Cdc25A is induced by c-myc .
Loss of TGF-beta mediated G1arrest in cancer
In nontransformed epithelial cells, TGF-β causes G1 arrest through downregulation of c-myc, inhibition of the G1 cdks and hypophosphorylation of pRb. Overlapping or redundant cell cycle controls assure growth arrest. In malignantly transformed cells, however, this redundancy is often lost and carcinoma-derived cells are usually refractory to growth inhibition by TGF-β . Indeed, in advanced cancers, TGF-β may promote tumour growth and metastatic progression . In this part of the discussion, we review how dysregulation of many different cell cycle mechanisms abrogate TGF-β arrest in cancer (Fig. 2).
Altered cdk inhibitor expression and function
Dysregulation of the INK4 family may contribute to TGF-β resistance in cancer. In human tumours, deletion of p15 often accompanies p16 deletion due to their proximity on chromosome 9p [72,73,74]. Silencing of p15 through promoter hypermethylation, which is observed in leukaemias, is associated with loss of TGF-β sensitivity [75,76]. In other TGF-β-resistant cells, however, p15 protein levels may increase normally, indicating that, at least in these lines, a functional p15 is not sufficient to mediate arrest by TGF-β .
Although p15 and p27 cooperate to inhibit the G1 cyclin-cdks in normal cells, neither of these cdk inhibitors are essential for G1 arrest by TGF-β. p15 is clearly not essential for TGF-β-mediated G1 arrest, because cells bearing p15 deletions can respond through upregulation of p21 and p27 [59,65], or downregulation of Cdc25A . Lymphocytes from p27-null mice can still arrest in response to TGF-β . Nonetheless, the requirement for p27 in arrest by TGF-β may differ in normal and transformed cells. Although inhibition of p27 expression through antisense p27 oligonucleotide transfection did not abrogate TGF-β-mediated arrest in finite lifespan HMECs, it did do so in breast cancer-derived lines (Donovan J, Slingerland J, unpublished data). In normal cells, multiple redundant pathways cooperate to mediate arrest, but in cancer cells the progressive loss of other checkpoints may make p27 indispensable for TGF-β-mediated arrest.
The antiproliferative role of p27 is frequently disrupted in human cancers. Although mutations in p27 are rare [78,79], accelerated proteolysis causes reduced p27 protein in many cancers, including breast, and may contribute to TGF-β resistance [37,80,81,82]. Less often, primary tumours may exhibit strong cytoplasmic p27 expression associated with poor prognosis. Cytoplasmic p27 has been observed in some advanced cancer-derived lines . Thus, some cancers may express a stable but inactivated p27. In a TGF-β-resistant HMEC line, we have observed stable cytoplasmic p27 localization, altered p27 phosphorylation and impaired binding of p27 to cyclin E-cdk2 (Ciarallo S, Slingerland J, unpublished data). The elucidation of how of mitogenic signalling pathways alter p27 inhibitor function may prove important insights into mechanisms of TGF-β resistance (see below).
Altered KIP function has also been observed in TGF-β-resistant prostate cancer cells. Although TGF-β caused an upregulation in p21-cdk2 binding, this kinase was not inhibited, suggesting that p21 may not function normally in these cells . Loss of p21 has also been observed in advanced breast cancers in association with a poor patient prognosis [85,86]. As for p27, functional inactivation of p21 could contribute to TGF-β resistance during breast cancer progression.
Cyclin overexpression and TGF-β resistance
Overexpression and/or amplification of the cyclin D1 gene is seen in up to 40% of breast cancers [87,88] and could contribute to TGF-β resistance. Indeed, cyclin D1 transfection of an oesophageal epithelial line conferred resistance to TGF-β . Increased cyclin E protein has also been observed in breast cancers [80,90]. Constitutive overexpression of cyclin E does not confer TGF-β resistance in Mv1Lu cells, however (Slingerland J, Reed S, unpublished data). Pathways that link impaired cyclin degradation with loss of cell cycle responses to TGF-β have yet to be elucidated.
Cdk4 gene amplification and activating mutation
Although loss of cdk4 does not contribute significantly to arrest by TGF-β because it occurs after most cells have entered G1 phase , ectopic cdk4 expression can abrogate TGF-β-mediated arrest . The increased cdk4 level may exceed titration by p15 and, in addition, sequestration of KIPs away from cyclin E-cdk2 into newly formed cyclin D-cdk4 complexes may lead to loss of cdk2 inhibition. Overexpression of cdk4 may contribute to TGF-β resistance in human cancers. Amplification of the cdk4 gene occurs in primary breast cancers  and dominant active cdk4 mutations have been observed in human malignant melanoma .
Activation of c-myc, and TGF-β resistance
TGF-β arrest-resistant cells often fail to downregulate c-myc . Moreover, oncogenic activation of c-myc, which is seen in a number of human malignancies, including breast cancer, may impair TGF-β responsiveness through a number of mechanisms.
Overexpression of c-Myc may increase G1 cyclin levels. c-Myc may regulate indirectly the expression of cyclins D1, E and A [52,53]. c-Myc induction of cyclin D1 or cyclin D2 may lead to the sequestration of p27 and p21 away from cyclin E-cdk2, and thus contribute to cyclin E-cdk2 activation [93,94]. These effects, however, which are best demonstrated in fibroblast lines, may not be relevant to TGF-β resistance in epithelial cells. In Mv1Lu cells, c-myc overexpression prevents arrest by TGF-β in part by inhibiting p15 induction . c-Myc effects on D-type cyclin expression and cyclin D-cdk4 complex formation were not sufficient to account for loss of the TGF-β response. Thus, repression of p15 by c-Myc may be important in the arrest-resistant phenotype.
Additional mechanisms link c-Myc with cyclin E-cdk2 activation. Overexpression of c-myc can induce a heat labile factor that binds p27 and inhibits its association with cyclin E-cdk2 . This effect is independent of p27 degradation. Although in some cell types cyclins D1 and D2 may be the c-myc-induced inhibitors of p27 [93,94], in other models the c-myc-induced inhibitor of p27 appears to be independent of D-type cyclins .
Oncogenic activation of c-myc may lead to Cdc25A overexpression and loss of TGF-β-mediated repression of Cdc25A . Overexpression of Cdc25A is observed in primary breast cancers and is associated with a poor patient prognosis (Loda M, personal communication). The increased Cdc25A may represent one of the checkpoints whose disruption makes subsequent disruption of p27 function more critical during breast cancer progression.
Activation of Ras and its effector pathways and TGF-β resistance
Overexpression of activated Ras has been shown to abrogate the antimitogenic effects of TGF-β . Mutational activation of ras is common in many human cancers and may be linked to TGF-β resistance through a number of mechanisms. Activated Ras can interfere with TGF-β signalling by altering Smad2 phosphorylation and signal transduction . Moreover, Ras activation can increase cyclin D1 levels through both transcriptional and posttranslational mechanisms [38,98,99]. Ras activation also accelerates p27 degradation [40,41], in some models requiring coexpression of Myc . Although ras mutations are not commonly observed in breast cancer, epidermal growth factor (EGF) and ErbB2 overexpression are, and both activate the Ras effector phosphatidylinositol-3 kinase . Oncogenic activation of different ras effector pathways may abrogate p27 function [41,101], contributing importantly to TGF-β resistance.
Regulation of other G1events by TGF-β
p53 may play a role in the TGF-β response in some cells. In murine keratinocytes, introduction of mutated p53 led to TGF-β resistance, and a correlation between p53 mutation and loss of responsiveness to TGF-β-mediated arrest has been observed in several cancers [102,103,104].
Constitutive expression of mdm2 can give rise to TGF-β resistance. Although the Mdm2 protein binds to p53 to mediate p53 proteolysis, the effects of Mdm2 on TGF-β sensitivity appear to be independent of p53 function, because expression of an Mdm2 mutant that failed to bind p53 also conferred resistance . Because overexpression of Mdm2 occurs in about 73% of breast cancers, this too may play a role in TGF-β resistance in vivo [85,106,107].
During the past decade the anatomy of cell cycle regulation has been 'worked out'. TGF-β-induced G1 arrest occurs through induction of p15 and p21 genes, repression of the c-myc, Cdc25A, cyclin E and cyclin A genes, and an increase in the association of p15, p21 and p27 with target cdks. Inactivation of G1 cyclin-cdks leads to pRb dephosphorylation and E2F inhibition. The discovery of the Smads as both transducers of TGF-β signalling and transcriptional regulators has been a major advance in this field. Mitogenic signalling via ras/mitogen-activated protein kinase has been shown to interfere with Smad activation . It will be of interest to ascertain whether cross-talk with Smads can also negatively regulate components of mitogenic signal transduction pathways. How growth factors and mitogenic pathways influence the transcriptional activation, intracellular localization and degradation of cyclins and cdk inhibitors is only beginning to be mapped. As these mechanisms are elucidated, we will be able to move from the myopic view of cyclin-cdk regulation in the nucleus, to a broader three-dimensional view of cell cycle regulation that encompasses extracellular and cytoplasmic signalling pathways. The next frontiers lie in the cytoplasm and at the gateway of the nuclear pore as we begin to elucidate how TGF-β/Smad signalling interfaces with transducers of mitogenic signals to regulate cyclin-cdk activities.
We thank members of the Slingerland laboratory for critical reading of the manuscript and Ms Sophie Ku for excellent secretarial assistance.
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