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1.  Small Molecule-Induced Mitochondrial Disruption Directs Prostate Cancer Inhibition via Unfolded Protein Response Signaling 
Oncotarget  2013;4(8):1212-1229.
We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy.
PMCID: PMC3787152  PMID: 23902736
Prostate cancer; cell cycle arrest; apoptosis; oxidative stress; small molecule inhibitor; mitochondrial function
2.  Glycovariant anti-CD37 monospecific protein therapeutic exhibits enhanced effector cell-mediated cytotoxicity against chronic and acute B cell malignancies 
mAbs  2013;5(5):723-735.
TRU-016 is a SMIPTM (monospecific protein therapeutic) molecule against the tetraspanin transmembrane family protein CD37 that is currently in Phase 2 trials in Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL). In an attempt to enhance the ADCC function of SMIP-016, the chimeric version of TRU-016, SMIP-016GV was engineered with a modification in a glycosylation site in the Fc domain. The wild-type and glycovariant SMIP proteins mediate comparable Type I antibody-like direct cytotoxicity in the presence of anti-human Fc crosslinker and show a similar tyrosine phosphorylation pattern post-treatment. However, NK cells stimulated with the SMIP-016GV exhibit enhanced activation and release 3-fold more interferon-γ compared with SMIP-016. SMIP-016GV shows enhanced ADCC function against cells expressing CD37 with NK cell effectors derived from both normal and CLL-affected individuals. Enhanced ADCC is observed against CLL cells and is sustained at concentrations of SMIP-016GV as low at 5E−6 µg/mL on cells expressing minimal CD37 antigen. In support of the biological relevance of this, SMIP-016GV mediates effective ADCC against primary acute lymphoblastic leukemia (ALL) cells with low surface expression of CD37. Collectively, these data suggest potential use of the novel therapeutic agent SMIP-016GV with enhanced effector function for B cell malignancies, including CLL and ALL therapy.
doi:10.4161/mabs.25282
PMCID: PMC3851225  PMID: 23883821
CD37; CLL; ALL; Protein Therapeutics
3.  The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cells 
BMC Cell Biology  2002;3:22.
Background
The cyclin-dependent kinase inhibitor p27 is a putative tumor suppressor that is downregulated in the majority of human prostate cancers. The mechanism of p27 down-regulation in prostate cancers in unknown, but presumably involves increased proteolysis mediated by the SCFSKP2 ubiquitin ligase complex. Here we used the human prostate cancer cell line LNCaP, which undergoes G1 cell cycle arrest in response to androgen, to examine the role of the SKP2 F-box protein in p27 regulation in prostate cancer.
Results
We show that androgen-induced G1 cell cycle arrest of LNCaP cells coincides with inhibition of cyclin-dependent kinase 2 activity and p27 accumulation caused by reduced p27 ubiquitylation activity. At the same time, androgen decreased expression of SKP2, but did not affect other components of SCFSKP2. Adenovirus-mediated overexpression of SKP2 led to ectopic down-regulation of p27 in asynchronous cells. Furthermore, SKP2 overexpression was sufficient to overcome p27 accumulation in androgen arrested cells by stimulating cellular p27 ubiquitylation activity. This resulted in transient activation of CDK2 activity, but was insufficient to override the androgen-induced G1 block.
Conclusions
Our studies suggest that SKP2 is a major determinant of p27 levels in human prostate cancer cells. Based on our in vitro studies, we suggest that overexpression of SKP2 may be one of the mechanisms that allow prostate cancer cells to escape growth control mediated by p27. Consequently, the SKP2 pathway may be a suitable target for novel prostate cancer therapies.
doi:10.1186/1471-2121-3-22
PMCID: PMC122093  PMID: 12188931
4.  Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA, and Skp2 
PLoS ONE  2014;9(10):e109170.
The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3AR cells partially blocked accumulation of p27Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.
doi:10.1371/journal.pone.0109170
PMCID: PMC4182885  PMID: 25271736
5.  Galbanic acid decreases androgen receptor abundance and signaling and induces G1 arrest in prostate cancer cells 
Androgen receptor (AR) signaling is crucial for the genesis and progression of prostate cancer (PCa). We compared the growth responses of AR(+) LNCaP and LNCaP C4-2 vs. AR(−) DU145 and PC-3 PCa cell lines to galbanic acid (GBA) isolated from the resin of medicinal herb Ferula assafoetida and assessed their connection to AR signaling and cell cycle regulatory pathways. Our results showed that GBA preferentially suppressed AR(+) PCa cell growth than AR(−) PCa cells. GBA induced a caspase-mediated apoptosis that was attenuated by a general caspase inhibitor. Subapoptotic GBA down-regulated AR protein in LNCaP cells primarily through promoting its proteasomal degradation, and inhibited AR-dependent transcription without affecting AR nuclear translocation. Whereas docking simulations predicted binding of GBA to the AR ligand binding domain with similarities and differences with the AR antagonist drug bicalutamide, LNCaP cell culture assays did not detect agonist activity of GBA. GBA and bicalutamide exerted greater than additive inhibitory effect on cell growth when used together. Subapoptotic GBA induced G1 arrest associated with an inhibition of cyclin/CDK4/6 pathway, especially cyclin D1 without the causal involvement of CDK inhibitory proteins P21Cip1 and P27Kip1. In summary, the novelty of GBA as an anti-AR compound resides in the distinction between GBA and bicalutamide with respect to AR protein turnover and a lack of agonist effect. Our observations of anti-AR and cell cycle arrest actions plus the anti-angiogenesis effect reported elsewhere suggest GBA as a multi-targeting drug candidate for the prevention and therapy of PCa.
doi:10.1002/ijc.25993
PMCID: PMC3137900  PMID: 21328348
Galbanic acid; Androgen receptor; Proteasomal degradation; Prostate cancer; G1 arrest
6.  High-Throughput Screening AlphaScreen Assay for Identification of Small-Molecule Inhibitors of Ubiquitin E3 Ligase SCFSkp2-Cks1 
Journal of biomolecular screening  2013;18(8):910-920.
Decreased levels of cell cycle inhibitor p27Kip1 due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27Kip1 expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27Kip1 expression could improve prognosis and prevent excessive cell proliferation. SCFSkp2 is one of the major ubiquitin E3 ligases responsible for degradation of p27Kip1. Ubiquitination of p27Kip1 also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27Kip1. It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27Kip1 degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27Kip1 ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27Kip1 ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.
doi:10.1177/1087057113485789
PMCID: PMC4168015  PMID: 23589337
E3 ligase; inhibitor; Skp2; Cks1; p27kip1; ubiquitin; proteolysis
7.  Inositol Hexaphosphate Inhibits Growth and Induces G1 Arrest and Apoptotic Death of Androgen-Dependent Human Prostate Carcinoma LNCaP Cells1 
Neoplasia (New York, N.Y.)  2004;6(5):646-659.
Abstract
Prostate cancer (PCA) is the most common invasive malignancy and the second leading cause of cancer-related deaths in the US male population. One approach to control this malignancy is its preventive intervention by dietary agents. Inositol hexaphosphate (IP6), a dietary constituent, has shown promising efficacy against various cancers; however, limited studies have been performed with IP6 against PCA. Here, we investigated the growth-inhibitory effect and associated mechanisms of IP6 in androgen-dependent human prostate carcinoma LNCaP cells. IP6 treatment of cells resulted in a strong growth inhibition and an increase in G1 cell population. In mechanistic studies, IP6 resulted in an increase in cyclin-dependent kinase inhibitors (CDKIs) Cip1/p21 and Kip1/p27 levels, together with a decrease in cyclin-dependent kinase (CDK) 4 and cyclin D1 protein levels. An increase in CDKI levels by IP6 also led to a concomitant increase in their interactions with CDK2 and CDK4, together with a strong decrease in the kinase activity of both CDKs. Downstream in CDKI-CDK-cyclin cascade, consistent with its inhibitory effect on CDK kinase activity, IP6 treatment of cells increased hypophosphorylated levels of retinoblastoma (Rb) with a decrease in Rb phosphorylation at serine 780, 807, and 811 sites, and caused a moderate to strong decrease in the levels of transcription factors E2F1, E2F4, and E2F5. In other studies, IP6 caused a dose- and a time-dependent apoptotic death of LNCaP cells, and a decrease in Bcl2 levels, causing a strong increase in Bax versus Bcl2 ratio, as well as an inhibition of constitutively active AKT phosphorylation. Taken together, these molecular alterations provide an insight into IP6-caused growth inhibition, G1 arrest, and apoptotic death of human prostate carcinoma LNCaP cells. Because early clinical PCA growth is an androgen-dependent response, the results of the present study employing androgen-dependent LNCaP cells suggest that IP6 has promise and potential to be effective against PCA.
PMCID: PMC1531669  PMID: 15548374
Inositol hexaphosphate; prostate cancer prevention; apoptosis; cell cycle; cyclin-dependent kinase inhibitor
8.  Thrombin Induces Tumor Cell Cycle Activation and Spontaneous Growth by Down-regulation of p27Kip1, in Association with the Up-regulation of Skp2 and MiR-222 
Cancer research  2009;69(8):3374-3381.
The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G0 and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27Kip1 and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27Kip1 with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27Kip1-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27Kip1, was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27Kip1.
doi:10.1158/0008-5472.CAN-08-4290
PMCID: PMC2680713  PMID: 19351827
9.  S-phase kinase-associated protein-2 (Skp2) promotes vascular smooth muscle cell proliferation and neointima formation in vivo 
Journal of Vascular Surgery  2009;50(5-4):1135-1142.
Objective
Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo.
Methods and Results
Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening.
Conclusion
This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.
Clinical Relevance
This manuscript describes our latest work investigating the role of the Skp2, an F-box protein component of the SCFskp2 ubiquitin-ligase, in promoting VSMC proliferation, and neointima formation in response to vascular injury in vivo. Our previous work has identified a major role for Skp2 as a key target for numerous positive and negative growth regulatory signals in vitro. These signals converge to regulate the expression of Skp2, which then controls cell-cycle progression by promoting degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Until now, there has been no data in the literature on the role played by Skp2 in the regulation of VSMC proliferation and neointima formation in vivo. Our current manuscript describes, we believe for the first time, the important role played by Skp2 in these processes, using both mouse and rat arterial injury models. This is important because proliferation of VSMCs underlies the development of postangioplasty or post-stenting restenosis, venous graft failure, and transplant arteriosclerosis. Our work demonstrates for the first time that Skp2 is a major regulator of VSMC proliferation and neointimal thickening in vivo in response to vascular injury and highlights Skp2 as a potential target for future strategies designed to combat vasculoproliferative diseases.
doi:10.1016/j.jvs.2009.07.066
PMCID: PMC2774860  PMID: 19878790
10.  3,3′-Diindolylmethane Induces a G1 Arrest in Human Prostate Cancer Cells Irrespective of Androgen Receptor and p53 Status 
Biochemical pharmacology  2009;78(5):469-476.
3,3′-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study we characterized the effect of DIM on cell cycle regulation in both androgen dependent LNCaP and androgen receptor negative-p53 mutant DU145 human prostate cancer cells. DIM had an antiproliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM mediated G1 cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased expression of the cell cycle inhibitor p27Kip1 protein in LNCaP and DU145 cells. Promoter deletion studies with p27Kip1 reporter gene constructs showed that this DIM-mediated increase in p27Kip1 was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27Kip1 and subsequent G1 arrest by DIM involves activation of the p38 MAPK pathway in the DU145 cells. Taken together, our results indicate that DIM is able to stop the cell cycle progression of human prostate cancer cells regardless of their androgen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle regulatory effect in DU145 cells.
doi:10.1016/j.bcp.2009.05.008
PMCID: PMC2706920  PMID: 19433067
3; 3′-Diinolylmethane; Prostate cancer; Cell cycle arrest; p27Kip1; p38 MAPK; Cancer
11.  Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization 
Cell cycle (Georgetown, Tex.)  2010;9(5):971-974.
F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G0 and during the G1 phase of the cell cycle, Skp2 is degraded via the APC/CCdh1 ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G0/G1 state. APC/CCdh1 binds Skp2 through an N-terminal domain (amino acids 46–94 in human Skp2). It has been shown that phosphorylation of Ser64 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/ PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCFSkp2 ubiquitin ligase, and Skp2 relocalization/ retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.
PMCID: PMC3827631  PMID: 20160477
Skp2; Akt; SCF; ubiquitin
12.  Mitogenic Signaling by the gep Oncogene Involves the Upregulation of S-Phase Kinase-Associated Protein 2 
Genes & cancer  2010;1(10):1033-1043.
The gep oncogene, defined by the activated mutant of the α-subunit of the G protein G12 (Gα12Q229L or Gα12QL), potently stimulates the proliferation of many different cell types in addition to inducing neoplastic transformation of several fibroblast cell lines. While it has been demonstrated that Gα12QL accelerates G1- to S-phase cell cycle progression, the precise mechanism through which Gα12 communicates to cell cycle machinery is largely unknown. In the present study, we report that the activated—mutational as well as receptor-mediated—Gα12 transmits its proliferative signals to cell cycle machinery by modulating the levels of the S-phase kinase-associated protein 2 (Skp2), an E3 ubiquitin ligase, involved in the regulation of the cyclin-dependent kinase inhibitor (CKI), p27Kip1. Our results show that the expression of Gα12QL leads to an increase in the levels of Skp2 with a correlatable decrease in p27Kip1 levels and subsequent increase in the activities of specific CDKs. By demonstrating that the transient expression of Gα12QL induces an increase in Skp2 levels with resultant downregulation of p27Kip1 in both NIH3T3 and human astrocytoma 1321N1 cells, we establish here that the effect of Gα12 on Skp2/ p27Kip1 is cell type independent. In addition, we demonstrate that LPA-stimulated proliferation and changes in Skp2 and p27Kip1 levels in 1321N1 cells could be inhibited by the expression of a dominant-negative mutant of Gα12, thereby pointing to the critical role of Gα12 in LPA-mediated mitogenic signaling. Our findings also indicate that LPA as well as Gα12-mediated upregulation of Skp2 requires a yet to be characterized mechanism involving JNK. Since Skp2 has been identified as an oncogene, and it is overexpressed in many cancers, our results presented here describe for the first time that Skp2 is a novel target in the cell cycle machinery through which Gα12 and its cognate receptors transmit their oncogenic signals.
doi:10.1177/1947601910390516
PMCID: PMC3083023  PMID: 21533006
[AQ: 1]
13.  Mitogenic Signaling by the gep Oncogene Involves the Upregulation of S-Phase Kinase-Associated Protein 2 
Genes & Cancer  2010;1(10):1033-1043.
The gep oncogene, defined by the activated mutant of the α-subunit of the G protein G12 (Gα12Q229L or Gα12QL), potently stimulates the proliferation of many different cell types in addition to inducing neoplastic transformation of several fibroblast cell lines. While it has been demonstrated that Gα12QL accelerates G1- to S-phase cell cycle progression, the precise mechanism through which Gα12 communicates to cell cycle machinery is largely unknown. In the present study, we report that the activated—mutational as well as receptor-mediated—Gα12 transmits its proliferative signals to cell cycle machinery by modulating the levels of the S-phase kinase-associated protein 2 (Skp2), an E3 ubiquitin ligase, involved in the regulation of the cyclin-dependent kinase inhibitor (CKI), p27Kip1. Our results show that the expression of Gα12QL leads to an increase in the levels of Skp2 with a correlatable decrease in p27Kip1 levels and subsequent increase in the activities of specific CDKs. By demonstrating that the transient expression of Gα12QL induces an increase in Skp2 levels with resultant downregulation of p27Kip1 in both NIH3T3 and human astrocytoma 1321N1 cells, we establish here that the effect of Gα12 on Skp2/p27Kip1 is cell type independent. In addition, we demonstrate that LPA-stimulated proliferation and changes in Skp2 and p27Kip1 levels in 1321N1 cells could be inhibited by the expression of a dominant-negative mutant of Gα12, thereby pointing to the critical role of Gα12 in LPA-mediated mitogenic signaling. Our findings also indicate that LPA as well as Gα12-mediated upregulation of Skp2 requires a yet to be characterized mechanism involving JNK. Since Skp2 has been identified as an oncogene, and it is overexpressed in many cancers, our results presented here describe for the first time that Skp2 is a novel target in the cell cycle machinery through which Gα12 and its cognate receptors transmit their oncogenic signals.
doi:10.1177/1947601910390516
PMCID: PMC3083023  PMID: 21533006
oncogene; G12; LPA; Skp2; JNK
14.  Upregulation of MircoRNA-370 Induces Proliferation in Human Prostate Cancer Cells by Downregulating the Transcription Factor FOXO1 
PLoS ONE  2012;7(9):e45825.
Forkhead box protein O1 (FOXO1), a key member of the FOXO family of transcription factors, acts as a tumor suppressor and has been associated with various key cellular functions, including cell growth, differentiation, apoptosis and angiogenesis. Therefore, it is puzzling why FOXO protein expression is downregulated in cancer cells. MicroRNAs, non-coding 20∼22 nucleotide single-stranded RNAs, result in translational repression or degradation and gene silencing of their target genes, and significantly contribute to the regulation of gene expression. In the current study, we report that miR-370 expression was significantly upregulated in five prostate cancer cell lines, compared to normal prostatic epithelial (PrEC) cells. Ectopic expression of miR-370 induced proliferation and increased the anchorage-independent growth and colony formation ability of DU145 and LNCaP prostate cancer cells, while inhibition of miR-370 reduced proliferation, anchorage-independent growth and colony formation ability. Furthermore, upregulation of miR-370 promoted the entry of DU145 and LNCaP prostate cancer cells into the G1/S cell cycle transition, which was associated with downregulation of the cyclin-dependent kinase (CDK) inhibitors, p27Kip1 and p21Cip1, and upregulation of the cell-cycle regulator cyclin D1 mRNA. Additionally, we demonstrated that miR-370 can downregulate expression of FOXO1 by directly targeting the FOXO1 3′-untranslated region. Taken together, our results suggest that miR-370 plays an important role in the proliferation of human prostate cancer cells, by directly suppressing the tumor suppressor FOXO1.
doi:10.1371/journal.pone.0045825
PMCID: PMC3445500  PMID: 23029264
15.  SCFSkp2 Complex Targeted by Epstein-Barr Virus Essential Nuclear Antigen 
Molecular and Cellular Biology  2005;25(5):1749-1763.
The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCFSkp2, known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCFSkp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCFSkp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCFSkp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCFSkp2-dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCFSkp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.
doi:10.1128/MCB.25.5.1749-1763.2005
PMCID: PMC549383  PMID: 15713632
16.  Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27Kip1 ubiquitination pathway in hepatocellular carcinoma 
Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.
doi:10.1038/emm.2014.23
PMCID: PMC4044675  PMID: 24854087
cell cycle; cytokinesis; hepatocellular carcinoma; KIF14; p27kip1; ubiquitination
17.  Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells 
PLoS ONE  2013;8(12):e82625.
Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.
doi:10.1371/journal.pone.0082625
PMCID: PMC3857776  PMID: 24349321
18.  Distinct in vitro binding properties of the anti-CD20 small modular immunopharmaceutical 2LM20-4 result in profound and sustained in vivo potency in cynomolgus monkeys 
Rheumatology (Oxford, England)  2011;50(6):1033-1044.
Objectives. To characterize the in vitro binding and effector function properties of CD20-directed small modular immunopharmaceutical (SMIP) 2LM20-4, and to compare its in vivo B-cell depletion activity with the mutated 2LM20-4 P331S [no in vitro complement-dependent cytotoxicity (CDC)] and rituximab in cynomolgus monkeys.
Methods. Direct binding is examined in flow cytometry, confocal microscopy, scatchard and lipid raft assays. Effector function assays include CDC and Fc-mediated cellular toxicity. In the 6-month-long in vivo B-cell depletion study, single i.v. dosages of 1 or 10 mg/kg of anti-CD20 proteins were administered to monkeys and B-cell counts were monitored in peripheral blood, bone marrow and lymph nodes.
Results. 2LM20-4 has lower saturation binding to human primary B cells and recruits fewer CD20 molecules into lipid rafts compared with rituximab; however, it induces higher in vitro CDC. In competitive binding, 2LM20-4 only partially displaces rituximab, suggesting that it binds to a fraction of CD20 molecules within certain locations of the plasma membrane as compared with rituximab. In monkeys, 2LM20-4 had more sustained B-cell depletion activity than rituximab in peripheral blood and had significantly more profound and sustained activity than 2LM20-4 P331S and rituximab in the lymph nodes.
Conclusions. SMIP 2LM20-4, which binds to a fraction of CD20 molecules as compared with rituximab, has more potent in vitro CDC, and more potent and sustained B-cell depletion activity in cynomolgus monkeys. Our work has considerable clinical relevance since it provides novel insights related to the emerging B-cell depletion therapies in autoimmune diseases.
doi:10.1093/rheumatology/keq423
PMCID: PMC3093928  PMID: 21258049
B-cell depletion; Autoimmune diseases; Anti-CD20 small modular immunopharmaceutical; In vitro properties; In vivo properties
19.  Skp2 directs Myc-mediated suppression of p27Kip1 yet has modest effects on Myc-driven lymphomagenesis 
Molecular cancer research : MCR  2010;8(3):353-362.
The universal cyclin-Cdk inhibitor p27Kip1 functions as a tumor suppressor and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E-Cdk2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the impact of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared to the effects of Cks1 loss. Collectively these findings suggest that Cks1 targets in addition to p27Kip1 are critical for Myc-driven proliferation and tumorigenesis.
doi:10.1158/1541-7786.MCR-09-0232
PMCID: PMC3095030  PMID: 20197382
Myc; Skp2; p27Kip1; lymphomagenesis
20.  Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements 
ACS chemical biology  2010;5(12):1169-1182.
An alternative strategy for inhibition of the cyclin dependent kinases in anti-tumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential of generating highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anti-cancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27KIP1, with cyclin D1. This information has been used shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 and which can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and non-peptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove which to date has not been carried out in a systematic fashion. Collectively, the data presented provides new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and anti-tumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.
doi:10.1021/cb1001262
PMCID: PMC3425359  PMID: 20843055
21.  Androgen Suppresses Proliferation of Castration-Resistant LNCaP 104-R2 Prostate Cancer Cells via Androgen Receptor, Skp2, and c-Myc 
Cancer science  2011;102(11):2022-2028.
Summary
Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoring testosterone level may suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets implant. 24 out of 33 tumors adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen (PSA). We then study the molecular mechanism lying underneath the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G1 cell cycle arrest via reduction of Skp2 and c-Myc, and induction of p27Kip1. 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with slower growing rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27Kip1. Nuclear AR and PSA expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype, while knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells via AR, Skp2, and c-Myc.
doi:10.1111/j.1349-7006.2011.02043.x
PMCID: PMC3200457  PMID: 21781227
22.  Specific Small Molecule Inhibitors of Skp2-Mediated p27 Degradation 
Chemistry & biology  2012;19(12):1515-1524.
SUMMARY
In the ubiquitin proteasome system, the E3 ligase SCF-Skp2 and its accessory protein Cks1 promote proliferation largely by inducing the degradation of the CDK inhibitor p27. Overexpression of Skp2 in human cancers correlates with poor prognosis, and deregulation of SCF-Skp2-Cks1 promotes tumorigenesis in animal models. We identified small molecule inhibitors specific to SCF-Skp2 activity using in silico screens targeted to the binding interface for p27. These compounds selectively inhibited Skp2-mediated p27 degradation by reducing p27 binding through key compound-receptor contacts. In cancer cells, the compounds induced p27 accumulation in a Skp2-dependent manner and promoted cell-type specific blocks in the G1 or G2/M phases. Designing SCF-Skp2 specific inhibitors may be a novel strategy to treat cancers dependent on the Skp2-p27 axis.
doi:10.1016/j.chembiol.2012.09.015
PMCID: PMC3530153  PMID: 23261596
23.  Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer 
PLoS ONE  2012;7(9):e46072.
The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA.
doi:10.1371/journal.pone.0046072
PMCID: PMC3459846  PMID: 23029392
24.  CDK9 Is Constitutively Expressed throughout the Cell Cycle, and Its Steady-State Expression Is Independent of SKP2 
Molecular and Cellular Biology  2003;23(15):5165-5173.
CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCFSKP2 ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G0, despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t1/2) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t1/2 of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins, such as p27, but did not affect the expression of endogenous CDK9. Ectopic overexpression of SKP2 led to reduction of p27 protein levels but had no effect on the expression of endogenous CDK9. Finally, downregulation of endogenous SKP2 gene expression by interfering RNA had no effect on CDK9 protein levels, whereas p27 protein levels increased dramatically. Therefore, the SCFSKP2 ubiquitin ligase does not regulate CDK9 expression in a cell cycle-dependent manner.
doi:10.1128/MCB.23.15.5165-5173.2003
PMCID: PMC165719  PMID: 12861003
25.  Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation 
Molecular Cancer  2011;10:129.
Background
We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.
Results
Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.
Conclusion
From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.
doi:10.1186/1476-4598-10-129
PMCID: PMC3212821  PMID: 21999842
prostate cancer; UCHL1; ubiquitin system; tumour suppression; signalling

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