The Role Of Cyclin D1 And PACSIN2 In Repressing Cellular Migration

Tumor is regarded as a cellular periodic disease; Tumorigenesis is associated with deregulation of proliferation, dysdifferentiation and the inhibition of apoptosis. Cyclin D, as a member of the Cyclin family of cell cycle regulators, may play an important role in cancer. While there are three D-type Cyclins, Cyclin D1, Cyclin D2 Cyclin D3, only Cyclin Dl is observed to be overexpressed frequently in cancer. Cyclin D1 is best known as the regulatory subunit of a dimeric holoenzyme including the cell cycle-dependent kinase CDK4/6, which phosphorylates and inactivates the retinoblastoma protein Rb to promote progression through the G1-S phase of the cell cycle. And in this manner promote cellular proliferation.The orderly transition through the cell cycle is orchestrated by the sequential activation and inactivation of Cyclin-dependent kinases. The regulatory subunits of the CDK4/6 kinase are encoded by the D-type Cyclins. Cyclin D1 is induced early during G1 phase progression by mitogenic stimuli, which activate both the transcription of Cyclin D1, through specific DNA sequences in the Cyclin D1 promoter, and through the assembly of complexes that require sustained mitogenic stimulation. The Cyclin D1/CDK complexes phosphorylate and inactivate the retinoblastoma (pRb) protein, and the NRF-1 (Nuclear respiratory factor 1) protein to induce nuclear DNA synthesis and inhibit mitochondrial metabolism respectively.In addition to its CDK-binding function, a lot of evidence now show that D-type Cyclins have CDK-independent properties. As previously proposed, the role of these properties in cellular growth, metabolism, and cellular differentiation are substantial. Cyclin Dl forms physical associations with more than 30 transcription factors or transcriptional coregulators. Cyclin D1 directly interacts with and represses or induces several different nuclear receptors and transcription factors, including the estrogen receptor (ER), androgen receptor (AR), peroxisome proliferator activated receptor gamma (PPARy), CCAAT enhance binding protein?(C/EBP?), and signal transducer and activator of transcription 3 (STAT3).Given its role in promoting cellular proliferation and modulating transcription, it is not surprising that Cyclin D1 is deregulated in cancer. Overexpression of the Cyclin D1 gene has been reported in a variety of human cancers including breast, colon, prostate, and hematopoietic malignancies. In invasive breast cancers, Cyclin D1 is overexpressed in up to 50% of cases. And in keeping with a role for Cyclin D1 in diverse human cancers, mice deficient in Cyclin D1 are resistant to oncogene-induced tumorigenesis. Also, Gastrointestinal tumors induced by mutation of the Apc gene are reduced in number by Cyclin D1 deficiency. Consistent with findings that Cyclin D1 antisense abrogates mammary epithelial cell growth induced by ErbB2, Cyclin D1-deficient mice are resistant to mammary gland tumorigenesis induced by Ras or ErbB2.Cyclin D1-deficient cells demonstrate reduced cellular survival and DNA synthesis increased mitochondrial size and activity and reduced cellular migration of diverse cell types including blood vessels, bone marrow macrophages, fibroblasts and mammary epithelial cells.Cell migration is essential for developmental morphogenesis, tissue repair, and tumor metastasis. Cancer cells spread from the initial site of tumor growth by first invading the surrounding tissue (migration), then entering the blood or lymph vessels (intravasation), and finally crossing the vessel wall to exit the vasculature (extravasation) in distal organs. The cancer cells then colonize the new site and proliferate to form a second tumor mass.Preventing metastasis presents an important therapeutic approach to cancer treatment. So as the first step of metastasis, learning more about the cellular different migration/invasion programmes will help us to understand how cancer cells disseminate and lead to new treatment strategies. By frequent overexpression of Cyclin D1 in cancer cells, new finding suggested that Cyclin D1 may have a central role in mediating invasion and metastasis of cancer cells by controlling Rho/ROCK signaling and matrix deposition of TSP-1. But how Cyclin D1 promotes cellular migration, we still do not know.A common polymorphism of the human Cyclin D1 gene has been associated with increased rate of cancer development. The polymorphism (A870G) is located at the splice donor region at the exon4-intron4 boundary and modulates the efficiency of alternate splicing. Alternate splicing results in distinct carboxyl terminal amino acid sequences. Characterization of the functional properties of the canonical Cyclin D1a and the alternate Cyclin D1b isoform has revealed each encodes subunits capable of phosphorylating pRb, but distinguishable abilities to regulate cellular migration. Cyclin D1a promotes migration of fibroblasts and mammary epithelial cells; however the Cyclin D1b is defective in promoting migration.It has been predicted the diverse functions regulated by Cyclin D1 in cellular differentiation, proliferation and migration may be governed by subpartitioning of Cyclin D1 into distinct subcellular compartments, or through physical association with distinct binding proteins. In this regard, Cyclin D1 associates with p160 coactivator SRC-1 to regulate estrogen receptor a activity. In association with transcription factors, Cyclin D1 regulates CDK-independent transcriptional activities of the androgen receptor, CEBP?and PPAR?.In view of the diverse functions of Cyclin D1 in DNA synthesis, oncogenesis, and migration, we hypothesized that Cyclin D1-associated proteins may mediate these functions by forming a complex with Cyclin D1.Thus, In order to determine adapter proteins regulating Cyclin D1 function, we immunopurified Cyclin D1-associated proteins by Mass spectrometry and sequence analysis and identified PACSIN2, original named chicken FAP52, which family members have been shown to function as cytoplasmic adapter proteins in focal adhesions, as Cyclin D1-associated proteins.In our study we will conduct the physical association between Cyclin D1 and PACSIN2, and the role of Cyclin Dl and PACSIN2 in the cellular migration.Part I PACSIN2 is Identified as a Cyclin D1-binding Protein and PACSIN2 can specifically Bind Cyclin D1 Through its DPF DomainObjective:To identify PACSIN2 as a Cyclin D1-binding protein, and investigate whether Cyclin D1 can specifically binding PACSIN2, rather than other family members, and the mechanism for this specific binding.Methods:1. Purification of Cyclin D1-associated proteins were analyzed by silver-stained gel, the associated proteins excised and in gel tryptic digest in analyzed by mass spectrometry2. Using IP-Westernblot to conduct the cell co-transfected with expression vector for Myc epitope-tagged PACSIN2 and Flag-tagged Cyclin Dl wild type or mutant expression vector.3. Using IP-Westernblot to conduct the cell co-transfected with PACSIN2 association with D-type Cyclin D1, including D1 Wild type or Mutant4. Using IP-Westernblot to conduct the cell co-transfected with Cyclin D1 association with PACSIN family, including PACSIN1,2,35. Identify the domain of PACSIN2 required for physical interaction with Cyclin D1, by using IP-Westernblot to conduct the cell co-transfected with C-terminus truncated mutant constructs of PACSIN2 together with Vector encoding flag-Cyclin D1.Results:1. Co-purifing proteins, include CDK4, Hsc70 and an additional protein was identified by mass spectrometry with sequence identified to the homolog of chicken52, now identified as PACSIN22. Delection of the acidic rich stretch of Cyclin D1 abrogated the binding to PACSIN2. Cyclin Dl and Cyclin D2, but not Cyclin D3 and Cyclin Dlb, co-precipitated with PACSIN2. 3. Immmunoprecipitation of Cyclin Dl was found to co-precipitated PACS1N2, but not PACSIN1 and PACSIN3.4. The deletion the amino acid residues from aa363 to aa382 abrogates the association of Cyclin D1 with PACSIN2Conclusion:1. Identification and confirmation of PACSIN2 as a Cyclin D1-binding protein2. Cyclin D1 binds to PACSIN2 through its C-terminal E-rich motif3. NPF domain of PACSIN2 is required for Cyclin D1 bindingPart?.PACSIN2 Represses Cellular Migration through Direct Association Cyclin Dla, but not the Cyclin DlbObjective:To find out the co-localization of Cyclin Dl and PACSIN2, Identify the biological function of PACSIN2 as a Cyclin D1-binding protein, and whether PACSIN2 acts it role depend on Cyclin D1 or not.Methods:1. Identify the co-localization of Cyclin D1 and PACSIN2 by Immunohistochemical straing of MRC5 cell2. Transwell migration assay in PACSIN2+/+ and PACSIN2-/- mouse skin fibroblast cell lines3. Transwell migration assay by Knocking down PACSIN2 with siRNA (h) in Prostate Cancer cell line LNCaP4. Cell spreading Assay by Knocking down PACSIN2 with siRNA (m) in Mouse embryonic fibroblast cell line NIH 3T3.5. Transwell migration assay by Knocking down PACSIN2 in Cyclin D1-/- cells, and Cyclin D1-/- cell transduced with either Cyclin D1a or Cyclin Dlb retrovirus.6. Cell spreading Assay by Knocking down PACSIN2 in Cyclin D1-/- cell transduced with either Cyclin Dla or Cyclin D1b retrovirus.Results:1. Cyclin D1 and PACSIN2 were co-localized in cell ruffle. 2. PACSIN2-/- mouse skin fibroblast cell migration ability increases, PACSIN2 siRNA (h) increases cellular migration in Prostate Cancer cell line LNCaP. 3. PACSIN2 siRNA (m) reduced cell spreading in Mouse embryonic fibroblast cell line NIH3T34. PACSIN2 siRNA increases cell migration in Cyclin-/-+Dla cell, but has no effection on Cyclin D1-/- and Cyclin-/-+Dlb cells5. PACSIN2 siRNA represses cell spreading in Cyclin-/-+Dla cell, but has no effection on Cyclin-/-+Dlb cell.Conclusion:1. PACSIN2 represses cellular migration both in normal cell and prostate cancer cell lines.2. Endogenous PACSIN2 enhances cell spreading via Cyclin D1a, and Cyclin Dla is required for PACSIN2 repression of cellular migration.So, we hypothesize Cyclin D1 protein binding partners that contribute to the diverse functions of Cyclin D1 such as governing DNA synthesis, contact-independent growth, angiogenesis, and cellular migration, are critical for understanding Cyclin D1 function. Through identifying polypeptides associated with Cyclin D1, by tandem mass spectrometry, the current studies have identified PACSIN2 as a new Cyclin D1-associated protein, which contributes to the induction of cellular migratory directionality. In the current study, Cyclin D1 colocalized with PACSIN2 at the leading edge of MRC5 cells, Endogenous PACSIN2 enhances cell spreading via Cyclin Dla, and PACSIN2 siRNA can increase cellular migration in prostate cancer cell, indicating a role for PACSIN2 in repressing cellular migration. Together with the requirement of Cyclin Dla for PACSIN2 to repress cellular migration and PACSIN2 association with cytoskeletal proteins, we conclude that PACSIN2 provides a likely candidate protein coordinating a subset of the cellular migratory functions of Cyclin D1, which leads to a better understanding of the complexes contributing to Cyclin D1-mediated cellular migration.