Alternate cyclin D1 mRNA splicing modulates P27<sup>KlP1</sup> binding and cell migration

نویسندگان

  • Zhiping Li
  • Thomas Jefferson
  • Chenguang Wang
  • Xuanmao Jiao
  • Sanjay Katiyar
  • Mathew C. Casimiro
  • George C. Prendergast
  • Michael J. Powell
  • Richard G. Pestell
چکیده

Cyclin D1 is an important cell cycle regulator but in cancer its overexpression also increases cellular migration mediated by p27 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N-terminus as the canonical cyclin D1a isoform but a distinct C-terminus. In this study we show that these different isoforms have unique properties with regard to the cellular migration function of cyclin D1. Whereas they displayed little difference in transcriptional co-repression assays on idealized reporter genes, microarray cDNA expression analysis revealed differential regulation of genes including those that influence cellular migration. Additionally, while cyclin D1a stabilized p27 KIP1 and inhibited RhoA-induced ROCK kinase activity, promoting cellular migration, cyclin D1b failed to stabilize p27 KIP1 or inhibit ROCK kinase activity and had no effect on migration. Our findings argue that alternate splicing is an important determinant of the function of cyclin D1 in cellular migration. The cyclin D1 gene was initially cloned as a breakpoint rearrangement in parathyroid adenoma (1). Subsequent studies confirmed increased cyclin D1 abundance in a variety of tumors including breast and gastrointestinal tumors (2). Molecular analysis demonstrated the cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein, pRb. Immunoneutralizing and antisense experiments revealed the requirement for cyclin D1 in progression through the early G1 phase of the cell cycle (reviewed in (2)). In addition to binding Cdks, cyclin D1 associates with several different intracellular proteins including the estrogen receptor (ERα), PPARγ, P/CAF (p300/CBP associated factor) (3,4) and the cyclin D1 myb-like binding protein (DMP1) (5-8). Cyclin D1 binds to ERα in breast cancer cells and overcomes the BRCA1-mediated repression of ERα activity (9,10). Homozygous deletion of the cyclin D1 gene results in mice with failure of terminal alveolar breast development during pregnancy, increased basal and UVinduced apoptosis, retinal apoptosis, defective angiogenesis, hepatic steatosis, and defective cellular migration (4,11-14). Previous epidemiological and clinical observations have demonstrated a correlation between cyclin D1 overexpression and cellular metastases (15). Molecular genetic analysis subsequently revealed a key role for cyclin D1 in cellular adhesion and migration (14). In particular, cyclin D1-deficient cells showed enhanced cellular adhesion and reduced migration into a wound, and genetic complementation experiments established a requirement for the CDK-binding but not the pRB-binding domain of cyclin D1 to rescue the cell migratory defect (16,17). Together, these studies demonstrated a key role for cyclin D1 in cell migration, including bone marrow-derived macrophages, fibroblasts, and mammary epithelial cells (14,16,17). Recently, a common polymorphism identified in the human cyclin D1 gene was found to be associated with an increased risk of cancer development (18-22). This polymorphism (A870G), located at the splice donor region at the exon 4-intron 4 boundary, modulates the efficiency of alternate splicing between exon 4 and 5. As a result of the altered splicing which occurs, the coding region downstream is altered such that the amino acid sequence of the carboxyl-terminus of cyclin D1 is altered (18). Thus, the two isoforms of cyclin D1 produced, the canonical isoform termed cyclin D1a and the alternately spliced isoform termed cyclin D1b, are identical in their N-termini but distinct in their C-termini. Notably, clinical studies have associated this polymorphism with an increased risk of colon and rectal cancer, early-onset squamous cell carcinoma, head and neck cancer, and transitional cell carcinoma of the bladder (23). Elegant in vitro analyses have demonstrated that the cyclin D1a and D1b isoforms have similar half lives in cultured cells, however, the cyclin D1b isoform is primarily nuclear in subcellular localization and relatively more resistant to phosphorylation-dependent nuclear export (24). Cyclin D1a blocks cells at the G1 phase whereas cyclin D1b blocks cells at the G0 phase in some (23), but not all studies (25). Notably, while both isoforms encode regulatory subunits, cyclin D1b has a reduced capacity to phosphorylate pRb (25,26). Yet despite serving as a less effective component of this pRb kinase, cyclin D1b conveys a greater transforming capacity which enhances colony formation of NIH3T3 cells. Thus, the basis for the enhanced transforming ability of cyclin D1b has been unclear. The Cdk inhibitor p27 KIP1 inhibits most cyclin/Cdk complexes, including cyclin E/Cdk2 and cyclin D/Cdk4 (27). In the majority of studies, reduced p27 KIP1 in tumors correlates with a poorer prognosis (28-31). p27 KIP1 plays an important role in regulating cellular migration through regulating either Rac (32), Rho GTPase activity (16,33) or Stathmin function (34). Mechanistic analyses presented here reveal that cyclin D1b evades binding to p27 KIP1 which may explain the increased cell transforming activity of this isoform. Furthermore, cyclin D1b lacks the pro-migratory function of cyclin D1a, implicating the C-terminal region and alternate splicing as important determinants of this

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تاریخ انتشار 2017