Cyclin D1 regulates the G1
to S progression of the cell cycle and is over expressed at the protein level in several cancers including ~50% of breast cancers, as seen by immunohistochemistry [1
]. In the G1
phase of the cell cycle mitogens induce cyclin D1 transcription resulting in an increase in levels of cytoplasmic cyclin D1 protein [2
]. The cyclin D1 protein binds to cyclin dependent kinase 4/6 (CDK4/6) forming a kinase complex that translocates into the nucleus. The cyclin D1/CDK4 complex, once activated, phosphorylates the tumor suppressor protein Rb, releasing it from the transcription factor E2F [3
]. E2F facilitates the transcription of genes necessary for progression through the S phase of the cell cycle, including cyclin E and cyclin A. Following a peak during the G1
phase, levels of cyclin D1 drop during the S phase of the cell cycle. This decrease has been attributed to cyclin D1 phosphorylation at Thr286, causing export from the nucleus via CRM-1 [5
]. Phosphorylation at Thr286 also promotes binding of cyclin D1 by the SCF complex and ubiquitination leading to degradation by the proteosome [6
]. GSK3β has been implicated as the major kinase in phosphorylation of cyclin D1 at Thr286 [8
]. However, GSK3β is active throughout the cell cycle and therefore cannot account for the cell cycle dependent change in cyclin D1 Thr286 phosphorylation. Furthermore, phosphorylation at Thr286 and proteosome degradation cannot fully account for cyclin D1 turnover. Wild type cyclin D1 has a half-life of 30 minutes, whereas a T286A mutant and a splice variant of cyclin D1 lacking the C-terminal tail and the Thr286 site (cyclin D1b), still are degraded but with slightly longer half-lives [9
]. Control of the cyclin D1 degradation pathway is still incompletely understood.
There is evidence that defects in cyclin D1 degradation might account for the cyclin D1 protein accumulation found in cancers. An in vitro
screen of 76 deubiquitinating enzymes showed that one particular enzyme, USP2, reacted with monoubiquitinated cyclin D1. Over expression of USP2 in 293 cells stabilized cyclin D1 protein levels by preventing ubiquitin-mediated proteasomal degradation. Knockdown of USP2 in HCT116 colorectal cancer, MCF-7 breast cancer, and PC-3 prostate cancer cells destabilized cyclin D1 and reduced cell proliferation [11
]. These results offer hope that approaches to control cyclin D1 protein levels by interfering with the degradation pathway might be effective in arresting tumor growth.
Phosphorylation of cyclin D1 induces its proteasomal degradation, therefore protein phosphatases have a putative role in dephosphorylation cyclin D1 to prevent its degradation. To date there is little information about the identity of the protein phosphatase responsible for dephosphorylation of cyclin D1. In yeast, levels of the G1
cyclins (CLN1, CLN2, and HCS26) that correspond to mammalian cyclin D1 are regulated in part by the Ser/Thr phosphatase Sit4 [12
]. In PC-3 human prostate cancer cells expression of a fusion protein of GFP with the non-catalytic N-terminus of the phosphatase PP6 (the human ortholog of Sit4) caused a G1
cell cycle arrest with a corresponding reduction in cyclin D1 levels [13
]. This response was not mimicked by expression of a PP2A N-terminal/GFP fusion protein. These results suggested a specific role for PP6 in regulation of cyclin D1 levels, whether by direct dephosphorylation or another mechanism is unknown.
Several toxins in nature have been found to potently inhibit the PPP family of Ser/Thr phosphatases that includes the type-2A phosphatases (PP2A, PP4 and PP6) and the type-1 phosphatase, PP1. These toxins include the polyketals okadaic acid and calyculin A, purified from marine sponges, cyclic peptides of the microcystin and nodularin groups produced by blue green algae, and the compound cantharidin, an epoxycyclohexane dicarboxylic anhydride produced by blister beetles. The toxins calyculin A, okadaic acid, and cantharidin were chosen for this study based on their cell permeability and chemical stability. These three toxins are reported to show preferential inhibition of type-2A phosphatases compared to type-1 phosphatases [14
]. The aim of this study was to examine whether inhibition of type-2A phosphatases would promote degradation of cyclin D1 levels in human breast cancer cells, exposing a mechanism by which small molecule inhibitors may aid in suppression of tumor cell proliferation. We found that treating MDA-MB-468, MDA-MB-231, and MCF-7 breast cancer cells with these phosphatase inhibitory toxins decreased levels of cyclin D1. Unexpectedly, calyculin A induced proteosome degradation of cyclin D1 in MDA-MB-468 cells at more than an order of magnitude lower doses than the other toxins. These very low doses of calyculin A (<10 nM) increased Ser/Thr phosphorylation of only a few recognized endogenous substrates of PPP phosphatases, suggesting differential inhibition of subcellular pools of type 2A phosphatases.