Stacy W. Blain

TGFβ Signaling in Growth Control, Cancer, and Heritable Disorders

We are grateful to members of the Massague laboratory for insightful discussions. R. S. L. would like to thank S. H. Roan for all her help. R. S. L is supported by an NIH Medical Scientist Training Program (MSTP) grant. S. W. B. is a Special Fellow of the Leukemia and Lymphoma Society. J. M. is an investigator of the Howard Hughes Medical Institute.

Differential Interaction of the Cyclin-dependent Kinase (Cdk) Inhibitor p27Kip1 with Cyclin A-Cdk2 and Cyclin D2-Cdk4

Although p27Kip1 has been considered a general inhibitor of G1 and S phase cyclin-dependent kinases, we report that the interaction of p27 with two such kinases, cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu cells containing a p27 inducible system, a 6-fold increase over the basal p27 level completely inhibited Cdk2 and cell cycle progression. In contrast, the same or a larger increase in p27 levels did not inhibit Cdk4 or its homologue Cdk6, despite extensive binding to these kinases. A p27-cyclin A-Cdk2 complex formed in vitro was essentially inactive, whereas a p27-cyclin D2-Cdk4 complex was active as a retinoblastoma kinase and served as a substrate for the Cdk-activating kinase Cak. High concentrations of p27 inhibited cyclin D2-Cdk4, apparently by conversion of active complexes into inactive ones by the binding of additional p27 molecules. In contrast to their differential interaction, cyclin A-Cdk2 and cyclin D2-Cdk4 were similarly inhibited by bound p21Cip1/Waf1. Roles of cyclin A-Cdk2 as a p27 target and cyclin D2-Cdk4 as a p27 reservoir may result from the differential ability of bound p27 to inhibit the kinase subunit in these complexes.

Myc downregulation by transforming growth factor beta required for activation of the p15(Ink4b) G(1) arrest pathway.

ABSTRACT The antimitogenic action of transforming growth factor β (TGF-β) in epithelial cells involves cyclin-dependent kinase (cdk) inhibitory gene responses and downregulation of c-Myc expression. Although the cdk inhibitory responses are sufficient for G1arrest, enforced expression of c-Myc prevents G1 arrest by TGF-β. We investigated the basis of this antagonism by using Mv1Lu lung epithelial cell lines that conditionally express levels of human c-Myc. We show that c-Myc prevents induction of the cdk4 inhibitor p15Ink4b and the subsequent inhibition of G1cdks by TGF-β. We assessed the significance of this effect by analyzing the oligomeric state of cdk4 in these cells. In proliferating cells, endogenous cdk4 is distributed among three populations: an abundant high-molecular-mass (>400-kDa) pool of latent cdk4 that serves as a source of cdk4 for cyclin D, a low-abundance pool containing active cyclin D-cdk4 complexes, and an inactive population of monomeric cdk4. Cell stimulation with TGF-β converts the latent and active cdk4 pools into inactive cdk4, an effect that is specifically mimicked by overexpression of p15 but not by other forms of G1 arrest. This process of TGF-β-induced cdk4 inactivation is completely blocked by expression of c-Myc, even though the latent and active cdk4 complexes from c-Myc-expressing cells remain sensitive to dissociation by p15 in vitro. c-Myc causes a small increase in cyclin D levels, but this effect contributes little to the loss of TGF-β responses in these cells. The evidence suggests that c-Myc interferes with TGF-β activation of the p15 G1arrest pathway. TGF-β must therefore downregulate c-Myc in order to activate this pathway.

p27 as a target for cancer therapeutics

We thank Kathryn Anderson for discussion regarding haploinsufficiency, and Joan Massague and Eric Holland for helpful discussion during the preparation of this manuscript.

Breast cancer banishes p27 from nucleus.

The cell-cycle inhibitor p27 is phosphorylated by the Akt kinase in breast cancer, according to three new studies. This phosphorylation keeps p27 in the cytoplasm and correlates with cancer aggressiveness (pages 1136–1144, 1145–1152 and 1153–1160.)

Differential Modification of p27Kip1 Controls Its Cyclin D-cdk4 Inhibitory Activity

ABSTRACT Whether p27 is a cyclin D-cdk4/6 inhibitor or not is controversial, and how it might switch between these two modes is unknown. Arguing for a two-state mechanism, we show that p27 bound to cyclin D-cdk4 can be both inhibitory and noninhibitory, due to its differential-growth-state-dependent tyrosine phosphorylation. We found that p27 from proliferating cells was noninhibitory but that p27 from arrested cells was inhibitory, and the transition from a bound noninhibitor to a bound inhibitor was not due to an increase in p27 concentration. Rather, two tyrosine residues (Y88 and Y89) in p27s cdk interaction domain were phosphorylated preferentially in proliferating cells, which converted p27 to a noninhibitor. Concordantly, mutation of these sites rendered p27 resistant to phosphorylation and locked it into the bound-inhibitor mode in vivo and in vitro. Y88 was directly phosphorylated in vitro by the tyrosine kinase Abl, which converted p27 to a cdk4-bound noninhibitor. These data show that the growth-state-dependent tyrosine phosphorylation of p27 modulates its inhibitory activity in vivo.

p27Kip1 Inhibits Cyclin D-Cyclin-Dependent Kinase 4 by Two Independent Modes

ABSTRACT Cell cycle progression is regulated by cyclin-dependent kinases (cdks), which in turn are regulated by their interactions with stoichiometric inhibitors, such as p27Kip1. Although p27 associates with cyclin D-cyclin-dependent kinase 4 (cdk4) constitutively, whether or not it inhibits this complex is dependent on the absence or presence of a specific tyrosine phosphorylation that converts p27 from a bound inhibitor to a bound noninhibitor under different growth conditions. This phosphorylation occurs within the 3-10 helix of p27 and may dislodge the helix from cdk4s active site to allow ATP binding. Here we show that the interaction of nonphosphorylated p27 with cdk4 also prevents the activating phosphorylation of the T-loop by cyclin H-cdk7, the cdk-activating kinase (CAK). Even though the cyclin H-cdk7 complex is present and active in contact-arrested cells, p27s association with cyclin D-cdk4 prevents T-loop phosphorylation. When p27 is tyrosine phosphorylated in proliferating cells or in vitro with the tyrosine Y kinase Abl, phosphorylation of cdk4 by cyclin H-cdk7 is permitted, even without dissociation of p27. This suggests that upon release from the contact-arrested state, a temporal order for the reactivation of inactive p27-cyclin D-cdk4 complexes must exist: p27 must be Y phosphorylated first, directly permitting cyclin H-cdk7 phosphorylation of residue T172 and the consequent restoration of kinase activity. The non-Y-phosphorylated p27-cyclin D-cdk4 complex could be phosphorylated by purified Csk1, a single-subunit CAK from fission yeast, but was still inactive due to p27s occlusion of the active site. Thus, the two modes by which p27 inhibits cyclin D-cdk4 are independent and may reinforce one another to inhibit kinase activity in contact-arrested cells, while maintaining a reservoir of preformed complex that can be activated rapidly upon cell cycle reentry.

Sustained proliferation in cancer: Mechanisms and novel therapeutic targets.

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression.

Different Sensitivity of the Transforming Growth Factor-β Cell Cycle Arrest Pathway to c-Myc and MDM-2

Recently, the oncoprotein MDM-2 was implicated in the transforming growth factor-β (TGF-β) growth inhibitory pathway by the finding that prolonged, constitutive expression of MDM-2 in mink lung epithelial cells could overcome the antiproliferative effect of TGF-β (Sun, P., Dong, P., Dai, K., Hannon, G. J., and Beach, D. (1998) Science 282, 2270–2272). However, using Mv1Lu cells conditionally expressing MDM-2, we found that MDM-2 does not overcome TGF-β-mediated growth arrest. No detectable changes were observed in various TGF-β responses, including cell cycle arrest, activation of transcriptional reporters, and TGF-β-dependent Smad2/3 nuclear accumulation. This finding was in direct contrast to the effect of forcing c-Myc expression, a bona fide member of the TGF-β growth inhibitory pathway, which renders cells refractory to TGF-β-induced cell cycle arrest. Our results suggest that an MDM-2-dependent increase in cell cycle progression may allow the acquisition of additional mutations over time and that these alterations then allow cells to evade a TGF-β-mediated growth arrest. Our conclusion is that, whereas c-Myc down-regulation by TGF-β is a required event in the cell cycle arrest response of epithelial cells, MDM-2 is not a direct participant in the normal TGF-β antiproliferative response.

S phase entry causes homocysteine-induced death while ataxia telangiectasia and Rad3 related protein functions anti-apoptotically to protect neurons

A major phenotype seen in neurodegenerative disorders is the selective loss of neurons due to apoptotic death and evidence suggests that inappropriate re-activation of cell cycle proteins in post-mitotic neurons may be responsible. To investigate whether reactivation of the G1 cell cycle proteins and S phase entry was linked with apoptosis, we examined homocysteine-induced neuronal cell death in a rat cortical neuron tissue culture system. Hyperhomocysteinaemia is a physiological risk factor for a variety of neurodegenerative diseases, including Alzheimers disease. We found that in response to homocysteine treatment, cyclin D1, and cyclin-dependent kinases 4 and 2 translocated to the nucleus, and p27 levels decreased. Both cyclin-dependent kinases 4 and 2 regained catalytic activity, the G1 gatekeeper retinoblastoma protein was phosphorylated and DNA synthesis was detected, suggesting transit into S phase. Double-labelling immunofluorescence showed a 95% co-localization of anti-bromodeoxyuridine labelling with apoptotic markers, demonstrating that those cells that entered S phase eventually died. Neurons could be protected from homocysteine-induced death by methods that inhibited G1 phase progression, including down-regulation of cyclin D1 expression, inhibition of cyclin-dependent kinases 4 or 2 activity by small molecule inhibitors, or use of the c-Abl kinase inhibitor, Gleevec, which blocked cyclin D and cyclin-dependent kinase 4 nuclear translocation. However, blocking cell cycle progression post G1, using DNA replication inhibitors, did not prevent apoptosis, suggesting that death was not preventable post the G1-S phase checkpoint. While homocysteine treatment caused DNA damage and activated the DNA damage response, its mechanism of action was distinct from that of more traditional DNA damaging agents, such as camptothecin, as it was p53-independent. Likewise, inhibition of the DNA damage sensors, ataxia-telangiectasia mutant and ataxia telangiectasia and Rad3 related proteins, did not rescue apoptosis and in fact exacerbated death, suggesting that the DNA damage response might normally function neuroprotectively to block S phase-dependent apoptosis induction. As cell cycle events appear to be maintained in vivo in affected neurons for weeks to years before apoptosis is observed, activation of the DNA damage response might be able to hold cell cycle-induced death in check.