Essential role for Rac in heregulin beta1 mitogenic signaling: a mechanism that involves epidermal growth factor receptor and is independent of ErbB4

Heregulins are a family of ligands for the ErbB3/ErbB4 receptors that play important roles in breast cancer cell proliferation and tumorigenesis. Limited information is available on the contribution of Rho GTPases to heregulin-mediated signaling. In breast cancer cells, heregulin beta1 (HRG) causes a strong activation of Rac; however, it does so with striking differences in kinetics compared to epidermal growth factor, which signals through ErbB1 (epidermal growth factor receptor [EGFR]). Using specific ErbB receptor inhibitors and depletion of receptors by RNA interference (RNAi), we established that, surprisingly, activation of Rac by HRG is mediated not only by ErbB3 and ErbB2 but also by transactivation of EGFR, and it is independent of ErbB4. Similar receptor requirements are observed for HRG-induced actin cytoskeleton reorganization and mitogenic activity via extracellular signal-regulated kinase (ERK). HRG-induced Rac activation was phosphatidylinositol 3-kinase dependent and Src independent. Furthermore, inactivation of Rac by expression of the Rac GTPase-activating protein beta2-chimerin inhibited HRG-induced ERK activation, mitogenicity, and migration in breast cancer cells. HRG mitogenic activity was also impaired by depletion of Rac1 using RNAi. Our studies established that Rac is a critical mediator of HRG mitogenic signaling in breast cancer cells and highlight additional levels of complexity for ErbB receptor coupling to downstream effectors that control aberrant proliferation and transformation.     

Biological effects of G1 phase arrest compound,sesquicillin, in human breast cancer cell lines

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.     

Cyclin D1 overexpression induces progestin resistance in T-47D breast cancer cells despite p27(Kip1) association with cyclin E-Cdk2

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.     

p21(Cip1) Promotes cyclin D1 nuclear accumulation via direct inhibition of nuclear export.

There is increasing evidence that p21(Cip1) and p27(Kip1) are requisite positive regulators of cyclin D1.CDK4 assembly and nuclear accumulation. Both Cip and Kip proteins can promote nuclear accumulation of cyclin D1, but the underlying mechanism has not been elucidated. We now provide evidence that p21(Cip1) promotes the nuclear accumulation of cyclin D1 complexes via inhibition of cyclin D1 nuclear export. In vivo, we demonstrate that p21(Cip1) can inhibit glycogen synthase kinase 3 beta-triggered cyclin D1 nuclear export and phosphorylation-dependent nucleocytoplasmic shuttling. Furthermore, we find that cyclin D1 nuclear accumulation in p21/p27 null cells can be restored through inhibition of CRM1-dependent nuclear export. The ability of p21(Cip1) to inhibit cyclin D1 nuclear export correlates with its ability to bind to Thr-286-phosphorylated cyclin D1 and thereby prevents cyclin D1.CRM1 association.     

Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression

Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.     

Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1).

Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G(1) cell cycle inhibitors p27(Kip1) and p21(Cip1) (Ckis) via a Myc-induced protein. We provide biochemical and genetic evidence to show that this sequestration is mediated via induction of cyclin D1 and/or cyclin D2 protein synthesis rates. Consistent with this conclusion, primary cells from cyclin D1(-/-) and cyclin D2(-/-) mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1(-/-) cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant forming kinase-defective, Cki-binding cyclin-cdk complexes. The sequestration function of D cyclins thus appears essential for Myc-induced cell cycle progression but dispensable for apoptosis.     

Effects of rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G(1) phase cyclin-dependent kinases

We recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase led to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1.     

Cell Death of MCF-7 Human Breast Cancer Cells Induced by EGFR Activation in the Absence of Other Growth Factors

The Epidermal Growth Factor (EGF) Receptor (EGFR) plays an important role in the growth and progression of breast cancer. Overexpression of EGFR or the high activity of EGFR signal pathway has been related with increases in cell proliferation and a poor prognosis in patients with breast cancer. Several human breast cancer cell lines depend on estrogen for their proliferation. EGF may bypass the requirement of estrogen for the proliferation of breast cancer cells. To evaluate this hypothesis, MCF-7 breast cancer cells were stimulated with EGF and the effects on cell proliferation, signal pathways, and cell cycle progression were determined. The results demonstrate that EGF stimulation in the absence of others growth factors induced a modest effect on cell proliferation and the induction of a cellular arrest in the G1 phase of the cell cycle. Although phosphorylation of AKT and ERK proteins were detected, this phosphorylation was insufficient to support of cell cycle progression. Cellular arrest in G1 phase was accompanied by an increase in p21CIP1 protein, down regulation of the BCL-2 protein, induction of caspase-8, and ARHI/NOEY2 an imprinted tumor suppressor gene. These results indicate that EGFR activation by itself is not sufficient for the proliferation of breast cancer cells and suggest the existence of a mechanism that induces apoptosis upon EGFR activation.     

p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor-induced vascular smooth muscle cell proliferation

The cyclin-dependent kinase inhibitors interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but these proteins have recently been shown to have positive regulatory effects on cyclin-cdk complex activity as well. Most of the previous work in this area has focussed on the finding that overexpressed p21(Waf1/Cip1) causes growth arrest. However, mice lacking p21(Waf1/Cip1) showed normal development with no aberrancy in their cell cycles, and antisense p21(Waf1/Cip1) has only been shown to prevent cell cycle arrest, leading to the conclusion that the cyclin kinase inhibitors may not be required for cell cycle progression. We found that transfection of several lines of vascular smooth muscle cells with antisense oligodeoxynucleotide specific to p21(Waf1/Cip1) correlates with decreased cyclin D1/cdk 4, but not cyclin E/cdk 2, association, yet, unexpectedly, results in dose-dependent inhibition of platelet-derived growth factor-BB-stimulated DNA synthesis and cell proliferation. Our finding that p21(Waf1/Cip1) exhibits permissive effects on growth factor-induced vascular smooth muscle cell cycle progression, such that its presence is required for growth factor-induced proliferation, is the first such report and opens up a fertile area of research relevant to diseases involving vascular cell proliferation.     

Insulin/insulin-like growth factor-I and estrogen cooperate to stimulate cyclin E-Cdk2 activation and cell Cycle progression in MCF-7 breast cancer cells through differential regulation of cyclin E and p21(WAF1/Cip1)

Estrogens and insulin/insulin-like growth factor-I (IGF-I) are potent mitogens for breast epithelial cells and, when co-administered, induce synergistic stimulation of cell proliferation. To investigate the molecular basis of this effect, a MCF-7 breast cancer cell model was established where serum deprivation and concurrent treatment with the pure estrogen antagonist, ICI 182780, inhibited growth factor and estrogen action and arrested cells in G(0)/G(1) phase. Subsequent stimulation with insulin or IGF-I alone failed to induce significant S-phase entry. However, these treatments increased cyclin D1, cyclin E, and p21 gene expression and induced the formation of active Cdk4 complexes but resulted in only minor increases in cyclin E-Cdk2 activity, likely due to recruitment of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/Cip1) into these complexes. Treatment with estradiol alone resulted in a greater increase in cyclin D1 gene expression but markedly decreased p21 expression, with a concurrent increase in Cdk4 and Cdk2 activity and subsequent synchronous entry of cells into S phase. Co-administration of insulin/IGF-I and estrogen induced synergistic stimulation of S-phase entry coincident with synergistic activation of high molecular mass (approximately 350 kDa) cyclin E-Cdk2 complexes lacking p21. To determine if the ability of estrogen to deplete p21 was central to these effects, cells stimulated with insulin and estradiol were infected with an adenovirus expressing p21. Induction of p21 to levels equivalent to those following treatment with insulin alone markedly inhibited the synergism between estradiol and insulin on S-phase entry. Thus the ability of estradiol to antagonize the insulin-induced increase in p21 gene expression, with consequent activation of cyclin E-Cdk2, is a central component of the synergistic stimulation of breast epithelial cell proliferation induced by simultaneous activation of the estrogen and insulin/IGF-I signaling pathways.     

Elevated expression of the estrogen receptor prevents the down-regulation of p21Waf1/Cip1 in hormone dependent breast cancer cells

Expression of an estrogen receptor alpha (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol-induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP-ER) hindered entry of estrogen treated MCF-7 cells into S phase of the cell cycle. In analyses of key cell-cycle regulating proteins, we observed that GFP-ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP-ER expressing cells but not in non-GFP-ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down-regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase.     

The ERK pathway involves positive and negative regulations of HT-29 colorectal cancer cell growth by extracellular zinc

Dietary zinc is an important trace element in the body and is related to both cell proliferation and growth arrest. A recent study found that extracellular zinc-sensing receptors trigger intracellular signal transduction in HT-29 human colorectal cancer cells. However, the signaling mechanism causing this growth regulation by extracellular zinc is not clearly understood. At 10- and 100-microM levels of ZnCl2 treatment, HT-29 cell growth and proliferation increased and decreased, respectively, in a minimally serum-starved medium (MSSM). A lack of significant increase in intracellular zinc levels after zinc treatment suggested that this differential growth regulation of HT-29 cells by extracellular zinc is acquired by receptor-mediated signal transduction. Moreover, this zinc-induced growth regulation was differentially affected by PD-98059, suggesting the involvement of the ERK pathway. Transient ERK activation and subsequent cyclin D1 induction were observed on adding 10 microM ZnCl2 in MSSM in the presence of cell proliferation. On the other hand, prolonged ERK activity was observed with a subsequent increase of cyclin D1 and p21(Cip/WAF1) on adding 100 microM ZnCl2 in MSSM, and this was associated with nonproliferation. Moreover, this ERK activation and cyclin D1 and p21(Cip/WAF1) induction were abolished by PD-98059 pretreatment. The differential regulations of cell growth, ERK activities, and cyclin D1 and p21(Cip/WAF1) inductions were also observed in serum-enriched medium containing higher zinc concentrations. Therefore, differential cell cycle regulator induction occurs by a common ERK pathway in the differential growth regulation of HT-29 cells by extracellular zinc.     

Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts

Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts that resembles the phenotype of scleroderma lung fibroblasts. We now demonstrate that PAR-1 expression is dramatically increased in lung tissue from scleroderma patients, where it is associated with inflammatory and fibroproliferative foci. We also observe that thrombin induces resistance to apoptosis in normal lung fibroblasts, and this process is regulated by protein kinase C (PKC)-epsilon but not by PKC-alpha. Overexpression of a constitutively active (c-a) form of PAR-1 or PKC-epsilon significantly inhibits Fas ligand-induced apoptosis in lung fibroblasts, whereas scleroderma lung fibroblasts are resistant to apoptosis de novo. Thrombin translocates p21Cip1/WAF1, a signaling molecule downstream of PKC, from the nucleus to cytoplasm in normal lung fibroblasts mimicking the localization of p21Cip1/WAF1 in scleroderma lung fibroblasts. Overexpression of c-a PKC-alpha or PKC-epsilon results in accumulation of p21Cip1/WAF1 in the cytoplasm. Depletion of PKC-alpha or inhibition of mitogen-activated protein kinase (MAPK) blocks thrombin-induced DNA synthesis in lung fibroblasts. Inhibition of PKC by calphostin or PKC-alpha, but not PKC-epsilon, by antisense oligonucleotides prevents thrombin-induced MAPK phosphorylation and accumulation of G(1) phase regulatory protein cyclin D1, suggesting that PKC-alpha, MAPK, and cyclin D1 mediate lung fibroblast proliferation. These data demonstrate that two distinct PKC isoforms mediate thrombin-induced resistance to apoptosis and proliferation and suggest that p21Cip1/WAF1 promotes both phenomena.     

Cooperation of p27(Kip1) and p18(INK4c) in progestin-mediated cell cycle arrest in T-47D breast cancer cells

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.