P27 Kip1 Protein Research Articles

Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1

Gap junctions and their structural proteins, connexins (Cxs), have been implicated in carcinogenesis. To explore the involvement of Cx32 in gastric carcinogenesis, immunochemical analysis of Cx32 and proliferation marker Ki67 using tissue-microarrayed human gastric cancer and normal tissues was performed. In addition, after Cx32 overexpression in the human gastric cancer cell line AGS, cell proliferation, cell cycle analyses, and p21Cip1 and p27Kip1 expression levels were examined by bromodeoxyuridine assay, flow cytometry, real-time RT-PCR, and western blotting. Immunohistochemical study noted a strong inverse correlation between Cx32 and Ki67 expression pattern as well as their location. In vitro, overexpression of Cx32 in AGS cells inhibited cell proliferation significantly. G1 arrest, up-regulation of cell cycle-regulatory proteins p21Cip1 and p27Kip1 was also found at both mRNA and protein levels. Taken together, Cx32 plays some roles in gastric cancer development by inhibiting gastric cancer cell proliferation through cell cycle arrest and cell cycle regulatory proteins. [BMB Reports 2013; 46(1): 25-30]

Expression of p27Kip1, a cell cycle repressor protein, is inversely associated with potential carcinogenic risk in the genetic rodent models of obesity and long-lived Ames dwarf mice

IntroductionThe association of genetic rodent models of obesity and cancer still remains a controversial issue. Although this controversy has largely been resolved in recent years for homozygous leptin receptor-deficient obese Zucker rats and homozygous long-lived Ames dwarf mice, it is still unresolved for homozygous leptin-deficient obese ob/ob mice. ObjectiveThe objective of the present study described below was to investigate whether the expression of the cell cycle repressor protein p27(Kip1) is (a) down-regulated in the tumor-free homozygous leptin receptor-deficient obese Zucker rats as well as tumor-free homozygous leptin-deficient obese ob/ob mice and (b) up-regulated in the tumor-free homozygous long-lived Ames dwarf mice. MethodsTo achieve this objective, we first performed western immunoblot analysis of the hepatic expression of p27. We then performed western immunoblot analysis and proteomic analysis of the hepatic expression of the proteins involved in the upstream molecular signaling pathways for the expression of p27. Lastly, we analyzed the serum levels of glucose, insulin, and branched-chain amino acids, all of which have been shown to regulate, causally and inversely, the expression of p27. Results/ConclusionsThe results indicated that the hepatic expression of p27 was down-regulated in the homozygous leptin receptor-deficient obese Zucker rats and up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the hepatic expression of p27 was down-regulated in the homozygous leptin-deficient obese ob/ob mice. This last observation was not completely consistent with all of the results of the published studies where homozygous leptin-deficient obese ob/ob mice were used.

Clinical significance of overexpressed cyclin-dependent kinase subunits 1 and 2 in esophageal carcinoma

The mammalian cyclin-dependent kinase subunit (Cks) family has two members, Cks1 and Cks2. Overexpression of Cks1 and Cks2 has been reported to be associated with high aggressiveness and poor prognosis in several malignancies, including prostate and hepatocellular carcinomas. However, whether Cks1 and Cks2 are overexpressed in esophageal carcinoma remains uncharacterized. To investigate whether overexpression of the Cks family is clinically relevant in esophageal carcinoma, and whether expression patterns of Cks1 and Cks2 can serve as biomarkers for esophageal carcinoma. Real-time quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot analyses were applied to detect the expression of Cks1 and Cks2 at the mRNA and protein levels, respectively. The associations between Cks1 or Cks2 expressions and clinical features and p27(kip1) expressions in esophageal carcinoma were analyzed. Comparing with the adjacent noncancerous tissues, esophageal carcinoma exhibited elevated expression of Cks1 in 58% cases at the mRNA level and 54% cases at the protein level, and elevated expression of Cks2 in 65% cases at the mRNA level and 61% cases at the protein level, respectively. The expressions of both Cks1 and Cks2 were negatively associated with the p27(kip1) protein level in the tumor tissues. Furthermore, overexpression of Cks1 and Cks2 in esophageal carcinoma was closely associated with poor pathological features of esophageal carcinoma, including higher histologic grade of tumor, regional lymph nodes invasion, and neoplastic embolus. Overexpression of Cks1 and Cks2 is associated with the aggressive tumor behaviors of esophageal carcinoma. Further efforts are needed to determine whether overexpression of Cks1 and Cks2 can serve as novel biomarkers for esophageal carcinoma.

Up-Regulation of p27Kip1 Protects hES Cells from Differentiation-Associated and Caspase 3-Dependent Apoptosis

Recently, it has been suggested that p27Kip1, the cell cycle regulatory protein, plays a pivotal role in the progression of normal differentiation in murine embryonic stem (mES) cells. In the current study, we investigated the role of p27Kip1 in the regulation of differentiation and apoptotic induction using Western blotting, quantitative real-time RT-PCR, and small interfering RNA (siRNA) assays and confocal laser scanning microscopic analysis of H9 human ES (hES) cells and H9-derived embryoid bodies (EBs) grown for 10 (EB10) and 20 days (EB20). Our results demonstrate that the proteins p27Kip1 and cyclin D3 are strongly associated with cellular differentiation, and, for the first time, show that up-regulation of p27Kip1 protects hES cells from inducing differentiation-associated and caspase 3-dependent apoptosis.

Causal co-expression method with module analysis to screen drugs with specific target

The considerable increase of investment in research and development by the pharmaceutical industry over the past three decades has not added the number of approved new drugs. An important issue ignored by drug discovery practice is the multi-dimensional interaction network between drugs and their targets. Thus, it is essential to view drug actions through the lens of network biology. In the current study, based on the co-expression network of transcription factors and their downstream genes, we proposed a novel approach, called causal co-expression method with module analysis, to screen drugs with specific target and fewer side effects. We presented a causal co-expression method with module analysis and it could be used in analyzing the microarray data of different drug candidates. At first, the differential wiring value (DW) was calculated to find some causal transcription factors (TFs) by combining with differential expression genes in the regulated networks. After the discovery of the causal TFs, co-expression module analysis method was applied to mine molecular pharmacology pathways around these causal TFs at molecular level. We applied our methods to two drug candidates, Argyrin A and Bortezomib, both with anti-cancer activities. We first obtained some differentially expressed transcription factors of cells treated with Argyrin A or Bortezomib. Nearly all these transcription factors are associated with the tumor suppressor protein p27kip1. Furthermore, module analysis showed that Bortezomib inhibited tumor growth not specifically by cell cycle and cell proliferation pathway, but through many basic metabolic processes which result in cell toxicity. In contrast, Argyrin A had influence on cell cycle, and was involved in DNA damage repair at the same time, showing that Argyrin A was a more suitable drug for anti-cancer treatment. Our study revealed that the causal co-expression method with module analysis was effective and can be used as a tool to evaluate drug candidates.

FOXO3a/p27kip1 expression and essential role after acute spinal cord injury in adult rat

FOXO3a (Forkhead Class box O3a), as an important direct target of the phosphatidylinositol 3-kinase (PI3K)/protein B (Akt) pathway, which regulates the cell survival and the cell-cycle progression. Recent reports showed that FOXO3a could inhibit cell-cycle progression at the G1/S transition by controlling transcription of the cyclin-dependent kinase inhibitor p27(kip1) , which is also a key regulator of the mammalian neurogenesis. To elucidate the expression and role of FOXO3a in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats, which showed a temporal-spatial expression pattern of FOXO3a. Temporally, FOXO3a protein level significantly reduced day 3 after injury, and following FOXO3a down-regulation, p27(kip1) protein and mRNA levels were also decreased after injury. Spatially, decreased levels of FOXO3a and p27(kip1) were predominant in astrocytes, which were regenerating axons and largely proliferated after injury. Furthermore in vitro, Western blot analysis, RT-PCR, and immunofluorescence staining analysis demonstrated the relationship between FOXO3a and p27(kip1) in primary astrocytes. FOXO3a modulated the cell cycle by transcriptional regulation of p27(kip1) in astrocytes. Administration of the PI3K pharmacological inhibitor LY294002 abrogated this effect by regulating FOXO3a and p27(kip1) expression and subcellular localization. These results suggest that decreased levels of FOXO3a and p27(kip1) in spinal cord are involved in axonal regeneration and the proliferation of glial cells after SCI.

Cyclin-Dependent Kinase 2 Controls Peripheral Immune Tolerance

Adaptive immunity requires signals from both the TCR and the costimulatory molecule CD28. These receptors activate multiple signaling pathways, including the cyclin-dependent kinase (CDK) cascade, and antigenic signals in the absence of costimulation result in a tolerant state that is enforced by the CDK inhibitory protein p27kip1. We find that CDK2, the major target of p27kip1, is highly active in T cells that infiltrate and reject cardiac allografts. We used mice genetically deficient for CDK2 to determine whether CDK2 is required for T cell alloimmunity. Blockade of CD28 costimulation alone was unable to inhibit the rejection of cardiac allografts by wild-type recipients. However, targeting this pathway in CDK2-deficient recipients led to long-term allograft survival. CDK2-deficient CD4(+) T cells proliferated normally in response to stimulation in vitro and in vivo, however, genetic, short hairpin RNA, or small molecule-mediated antagonism of CDK2 resulted in decreased production of IL-2 and IFN-γ. In addition, surviving grafts from CDK2-deficient recipients showed increased infiltration of Foxp3(+) regulatory T cells (Treg), and Treg from CDK2-deficient mice exhibited increased suppressive activity in vitro and in an in vivo model of inflammatory bowel disease. These data suggest that p27kip1 promotes peripheral tolerance through its ability to inhibit CDK2, which otherwise acts to promote conventional T cell differentiation and restrict Treg function.

Clinical Implications of p57KIP2Expression in Breast Cancer

To study the relationship between expression of p57KIP2 and prognosis and other clinicopathological parameters in invasive breast cancers. We assessed the expression of p57KIP2 in 89 cases of invasive breast cancer and 20 cases of normal breast tissue by immunohistochemical methods and analyzed the results with SPSS software (ver. 16.0). The positive expression rates of p57KIP2 protein in the invasive breast cancers and surrounding normal tissue were 30.3% (27/89) and 65% (13/20), respectively. Cases with no p57KIP2 expression exhibited a significantly higher post-operative distant metastasis rate than those with p57KIP2 expression (37.9% vs. 14.8%; P = 0.01). DFS analysis showed that p57KIP2-/C-erbB-2μ tumors also exhibited a significantly higher post-operative distant metastasis rate than the other groups (66.7% vs. 29.2%; P = 0.007), as did p57KIP2-/p53μ tumors (64.3% vs. 22.7%; P = 0.001). Survival analysis revealed that p57KIP2 was associated with breast cancer-specific survival overall (P = 0.045, log-rank test). Subgroup analysis demonstrated that individuals with p57KIP2-/C-erbB-2μ tumors experienced significantly worse post-operative survival than those with p57KIP2- /C-erbB-2- or other tumors (P = 0.006, log-rank test). p57KIP2-/p53μ tumors were associated with significantly worse post-operative survival than p57KIP2-/p53- or other tumors (P = 0.001, log-rank test). Cox regression analysis showed that p57KIP2 was a non-independent prognostic factor for breast cancer (P = 0.303). p57KIP2 is expressed at low levels in invasive breast cancer and is associated with better overall survival rate and disease-free survival in breast cancer patients, but it was a non-independent prognostic factor for breast cancer. Thus, the connection between p57KIP2/p53 and p57KIP2/C-erbB-2 may provide biomarkers for breast cancer.

Oncogenic Role of Skp2 and p27Kip1 in Intraductal Proliferative Lesions of the Breast

To investigate whether the connection of p27(Kip1) to S-phase kinase-associated protein 2 (Skp2) plays an oncogenic role in intraductal proliferative lesions of the breast. Here we investigated the mechanism involved in association of Skp2’s degradation of p27(Kip1) with the breast carcinogenesis by immunohistochemical method through detection of Skp2 and p27(Kip1) protein levels in 120 paraffin-embedded tissues of intraductal proliferative lesions including usual ductal hyperplasia (UDH, n=30), atypical ductal hyperplasia (n=30), flat epithelial atypia (FEA, n=30), and ductal carcinoma in situ (DCIS, n=30). Moreover, the expression status of Skp2 and p27(Kip1) in 30 cases of the normal breast paraffin-embedded tissues were explored. The DCIS group was with the highest Skp2 level and the lowest p27(Kip1) level, and the UDH group was with the lowest Skp2 level and the highest p27(Kip1) level.Both Skp2 and p27(Kip1) levels in the DCIS group were significantly different from those in the UDH group (all P<0.01).The levels of Skp2 and p27(Kip1) in the FEA group were significantly different from both the DCIS and UDH groups (all P<0.05).p27(Kip1) was negatively correlated with Skp2 in both the UDH group (r=-0.629, P=0.026) and DCIS group (r=-0.893, P=0.000). Overexpression of Skp2 might be the mechanism underlying p27(Kip1) over degradation.

99 TANSHINONE IIA ATTENUATED HYPOXIA-INDUCED PULMONARY HYPERTENSION VIA UPGRADING KV CHANNELS AND P27KIP1

Objectives: Pulmonary hypertension induced by hypoxia is the characterized by hypoxic pulmonary vasoconstriction (HPV) and pulmonary vascular structure remodeling (PVSR). Tanshinone IIA (TIIA) has numerous biological protective effects. Purpose of the paper is to investigated whether KV channels and P27kip1 were responsible for the protective effect of TIIA on HPV and PVSR. Basic procedures and methods: Effects of TIIA on intrapulmonary arteriole (IPA) rings and the IKV currents of pulmonary artery smooth muscle cells (PASMCs) isolated from rats in acute hypoxia experiments and chronic hypoxia experiments were determined. Main findings: results showed that TIIA attenuated acute hypoxia-induced IPA rings vasoconstriction which may be partially through recovering the down- regulation of IKV currents in PASMCs. TIIA markedly alleviated the increased weight of right ventricle/left ventricle plus septum ratio, medial width of pulmonary arterioles, decreased expression of Kv2.1 and Kv1.5 and the down-regulation of IKV currents induced by chronic hypoxia. In addition, TIIA increased the p27kip1 protein amount, but did not affect the p27kip1 mRNA amount. TIIA kept p27kip1 from being degraded through repressing the activity of Akt and decreasing the production of Skp-2. Conclusions: Principle conclusions: these data demonstrates that TIIA significantly attenuated acute and chronic hypoxia-induced HPV and PVSR, which were probably through up-regulation KV channels and p27kip1.

Successive Phosphorylation of p27KIP1 Protein at Serine-10 and C Terminus Crucially Controls Its Potency to Inactivate Cdk2

During the G(1)-S transition, the activity of Cdk2 is regulated by its association with p27(KIP1), which in rodent fibroblasts undergoes phosphorylation mainly at serine 10, threonine 187, and C-terminal threonine 197 by KIS, Cdk2, and Pim or ROCK, respectively. Recently Cdc6 the AAA+ ATPase, identified initially to assemble pre-replicative complexes on origins of replication and later to activate p21(CIP1)-inactivated Cdk2, was found also to activate p27-bound Cdk2 but only after the bound p27 is C-terminally phosphorylated. On the other hand, the biological significance of the serine 10 phosphorylation remains elusive aside from its involvement in the stability of p27 itself. We report here that serine 10 phosphorylation is required for efficient C-terminal phosphorylation of its own by PIM and ROCK kinases and critically controls the potency of p27 as a Cdk2 inhibitor. In vitro, PIM1 and active ROCK1 efficiently phosphorylated free as well as Cdk2-bound p27 but only when the p27 was phosphorylated at Ser-10 in advance. Consistently, a Ser-10 nonphosphorylatable mutant p27 protein was not phosphorylated at the C terminus in vivo. Furthermore, when double-phosphorylated, free p27 was no longer a potent inhibitor of Cdk2, and Cdk2-bound p27 could be removed by Cdc6 to reactivate the Cdk2. Thus, phosphorylation at these two sites crucially controls the potency of this CDK inhibitor in two distinct modes.

P27kip1 Protein Levels Reflect a Nexus of Oncogenic Signaling during Cell Transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.

Over-expression of BMPR-IB reduces the malignancy of glioblastoma cells by upregulation of p21 and p27Kip1

BackgroundIn our previous study, we detected decreased expression of phospho-Smad1/5/8 and its upstream signaling molecule, bone morphogenetic protein receptor IB subunit (BMPR-IB), in certain glioblastoma tissues, unlike normal brain tissues. In order to clarify the functional roles and mechanism of BMPR-IB in the development of glioblastoma, we studied the effects of BMPR-IB overexpression on glioblastoma cell lines in vitro and in vivo.MethodsWe selected glioblastoma cell lines U251, U87, SF763, which have different expression of BMPR-IB to be the research subjects. Colony formation analysis and FACS were used to detect the effects of BMPR-IB on the growth and proliferation of glioblastoma cells in vivo. Immunofluresence was used to detect the differentiation changes after BMPR-IB overexpression or knocking-down. Then we used subcutaneous and intracranial tumor models to study the effect of BMPR-IB on the growth and differentiation of glioblastoma cells in vivo. The genetic alterations involved in this process were examined by real-time PCR and western blot analysis.ed.Results and conclusionForced BMPR-IB expression in malignant human glioma cells, which exhibit lower expression of BMPR-IB, induced the phosphorylation and nuclear localization of smad1/5/8 and arrested the cell cycle in G1. Additionally, BMPR-IB overexpression could suppress anchorage-independent growth and promote differentiation of theses glioblastoma cells. Furthermore, overexpression of BMPR-IB inhibited the growth of subcutaneous and intracranial tumor xenografts and prolonged the survival of mice injected intracranially with BMPR-IB-overexpressing glioblastoma cells. Conversely, inhibition of BMPR-IB caused SF763 malignant glioma cells, a line known to exhibit high BMPR-IB expression that does not form tumors when used for xenografts, to show increased growth and regain tumorigenicity in a nude mouse model system, ultimately shortening the survival of these mice. We also observed significant accumulation of p21 and p27kip1 proteins in response to BMPR-IB overexpression. Our study suggests that overexpression of BMPR-IB may arrest and induce the differentiation of glioblastoma cells due to upregulation of p21 and p27kip1 in vitro and that in vivo and decreased expression of BMPR-IB in human glioblastoma cells contributes to glioma tumorigenicity. BMPR-IB could represent a new potential therapeutic target for malignant human gliomas.

Abstract 3057: CDK5 modulates the cell cycle, apoptosis and paclitaxel sensitivity in human ovarian cancer cells with wild-type p53 function

Abstract Cyclin-dependent kinase 5 (CDK5) is a ubiquitously expressed serine/threonine kinase that was initially identified in bovine brain extracts and plays an important role in normal neuron development and neurodegenerative diseases. This molecule has recently been shown to possess many cellular functions in non-neuronal tissues as well. Using a kinome siRNA screen, we identified CDK5 as a kinase that modulates paclitaxel sensitivity in human ovarian cancer cells. The aim of this report is to identify mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancer cells. Despite nucleotide sequence homology with human CDK1, a role of CDK5 in regulating the cell cycle remains unclear. Here we demonstrate dual roles of CDK5 in regulating the cell cycle and apoptosis. We have found that knockdown of CDK5 with multiple siRNAs markedly inhibits cell proliferation and increases paclitaxel sensitivity in several p53 wild type ovarian cancer cell lines using assays for cell viability and clonogenic growth. CDK5 knockdown with or without paclitaxel treatment induces G1 arrest of the cell cycle and apoptotic cell death linked to a significant induction of p53, p21Cip1 and p27Kip1 proteins. Induction of G1 arrest following CDK5 knockdown depends on induction of p21Cip1 and p27Kip1, since silencing p21Cip1 and/or p27Kip1 dramatically reduces CDK5 knockdown-induced G1 arrest. A CDK5 knockdown-induced increase of p53 expression contributes both to CDK5 knockdown-induced G1 arrest and to apoptosis, as silencing p53 markedly diminishes CDK5 knockdown-induced G1 arrest and apoptosis. As expected, a CDK5 knockdown-induced increase in p21Cip1 expression depends on p53-mediated transcriptional activation demonstrated by QRT-PCR and reporter luciferase assays. CDK5 knockdown activates caspase-3 and a pan-caspase inhibitor completely blocks CDK5 knockdown-induced apoptosis. Significant Inhibition of Bcl-2 expression is found in CDK5 knockdown-induced apoptosis and additional inhibition of Bcl-2 expression is found with combined CDK5 and paclitaxel treatment. Taken together, these data suggest that CDK5 can modulate paclitaxel sensitivity through regulating both the cell cycle and apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells with wild-type p53 function, providing a novel approach for enhancing the efficacies of paclitaxel-based regimens for low grade ovarian cancer in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3057. doi:1538-7445.AM2012-3057

Abstract 3040: Terbinafine inhibits oral squamous cell carcinoma growth through anti-cancer cell proliferation and anti-angiogenesis

Abstract ABSTRACT Terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, has been reported to exert an anti-tumor effect in various cancer cells. However, the effect of TB on oral cancer has not been evaluated. Herein we demonstrated that TB (0-60 μM) concentration-dependently decreased cell number in cultured human oral squamous cell carcinoma (OSCC), KB cells. The anti-proliferation effect of TB was also observed in two other OSCC cell lines, SAS and SCC 15. TB (60 μM) was not cytotoxic and its inhibition on KB cell growth was reversible. [3H]thymidine incorporation and flow cytometric analyses revealed that TB inhibited DNA synthesis and induced the G0/G1 cell cycle arrest. The TB-induced cell cycle arrest occurred when the cyclin-dependent kinase 2 (CDK2) activity was inhibited just as the protein levels of p21cip1 and p27kip1 were increased. The TB-induced G0/G1 cell cycle arrest was completely blocked when the expressions of p21cip1 and p27kip1 were knocked-down together. Taken together, these results suggest that the p21cip1- and p27kip1-associated signaling pathways might be involved in the TB-induced anti-proliferation in KB cells. In vivo, TB (50 mg/kg, i.p.) significantly inhibited the KB tumor size. In these TB-treated tumors, increases in the levels of p21cip1 and p27kip1 protein and decreases in the number of proliferating cell nuclear antigen (PCNA)-positive cells and the microvessel density (MVD) were observed. These findings demonstrate for the first time that TB might have potential to serve as a therapeutic tool in the treatment of oral cancer. 1 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3040. doi:1538-7445.AM2012-3040

The Effects of Combined Treatment with an HMG-CoA Reductase Inhibitor and PPARγ Agonist on the Activation of Rat Pancreatic Stellate Cells

Background/AimsHydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) and peroxisome proliferator-activated receptor gamma (PPARγ) ligands can modulate cellular differentiation, proliferation, and apoptosis through various pathways. It has been shown that HMG-CoA reductase inhibitors and PPARγ agonists separately inhibit pancreatic stellate cell (PaSC) activation. We studied the effects of a combination of both types of drugs on activated PaSCs via platelet-derived growth factor (PDGF), which has not previously been reported. The present study was performed to elucidate the underlying mechanisms of these effects by focusing on the impact of the signaling associated with cell-cycle progression.MethodsPrimary cultures of rat PaSCs were exposed to simvastatin and troglitazone. Proliferation was quantified using the BrdU method, and cell-cycle analysis was performed using a fluorescent activated cell sorter. The protein expression levels of smooth muscle actin (SMA), extracellular signal-regulated kinase (ERK), and a cell cycle machinery protein (p27Kip1) were investigated using Western blot analysis.ResultsSimvastatin reversed the effects of PDGF on cell proliferation in a dose-dependent manner. The combination of a low concentration of simvastatin (1 mM) and troglitazone (10 mM) synergistically reversed the effects of PDGF on cell proliferation but had no effect on cell viability. The expression of a-SMA was markedly attenuated by combining the two drugs, which blocked the cell cycle beyond the G0/G1 phase by reducing the levels of phosphorylated ERK and reversed the expression of p27Kip1 interrupted by PDGF.ConclusionsSimvastatin and troglitazone synergistically inhibited cell proliferation in activated PaSCs by blocking the cell cycle beyond the G0/G1 phase. This inhibition was due to the synergistic modulation of the ERK pathway and the cell cycle machinery protein p27Kip1.

Abstract 3808: Tetrandrine blocks cell cycle progression and promotes growth arrest in prostate cancer cells by up-regulating CDK inhibitors (p21 and p27) and suppressing cyclin expression.

Abstract Introduction: Prostate cancer (PCa) is the second leading cause of cancer deaths in men. De-regulated cell cycle progression is one of the hallmarks of cancer cells. While many agents are available to inhibit growth and proliferation of normal cells, we do not have efficient agents that inhibit growth and proliferation of tumor cells, and no agents are able to achieve growth arrest and inhibition of cell cycle progression in castrate resistant prostate cancer. In this study we evaluated effects of Tetrandrine (Tet: a bis-benzylisoquinoline alkaloid isolated from the root of Stephania tetrandra) that has been shown to have diverse biological properties, on cell growth, proliferation and cell cycle progression in prostate cancer cells. Methods: Prostate cancer cells, LNCaP cells (androgen dependent), LNCaP C-4 and C4-B (castrate resistant lineages derived from LNCaP cells) cells were obtained from ATTCC. Cells were maintained in select media and grown in 5% CO2 in a humidified incubator maintained at 37C. Where indicated Tet (1-20 uM) was added to the growth media for various time points as indicated. Cell growth and proliferation was monitored by MTT assay and cell cycle distribution was measured by FACS assay following labeling of the cells with Propedium Iodide. Western Blotting was used to monitor changes in the levels of various cell cycle related proteins. Results: Tet treatment inhibited cell growth and proliferation in all three cell lines tested. The effects of Tet on cell growth and proliferation were dose dependent with complete growth inhibition at 20 uM. Tet treatment resulted in a significant increase in cyclin dependent kinase inhibitory (CDKi) protein p21 (WAF1, Cip1) and p27 (Kip1) and with p21 increase being most prominent. Moreover, p21 was associated with Cyclin D kinases as monitored by IP assays. Gene expression analysis revealed that Tet decreased mRNA levels of cyclins. FACS analysis revealed that Tet treatment resulted in cell cycle arrest in G1/G0 phase of cell cycle. Finally TET inhibited growth of LNCaP-derived tumors in nude mice. These in vivo effects of TET were also associated with a dramatic increase in p21. Conclusions: Tet inhibits growth and proliferation of prostate cancer cells by effectively blocking cell cycle progression, in part by mediating increase in p21 (WAF1, Cip1) and p27 (Kip1) proteins and suppressing cyclin expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3808. doi:1538-7445.AM2012-3808

Abstract 4207: CCN5/WISP-2 upregulates p27Kip1 in breast cancer cell lines through AKT/FOXO3a signaling

Abstract CCN5 is a member of the CCN (cysteine rich 61-connective tissue growth factor-nephroblastoma-overexpressed) family of growth factors. Our studies have shown that CCN5 is mainly expressed in less aggressive human breast cancer cells (i.e., MCF-7 and ZR-75-1) and noninvasive lesion (i.e., atypical ductal hyperplasia and ductal carcinoma in situ), whereas its expression is minimally detected in moderately aggressive breast cancer cell lines (i.e., SKBR-3), and it is undetected in triple negative highly aggressive breast cancer cell line (i.e., MDA-MB-231) and invasive lesion. Ectopic expression of CCN5 has been shown to inhibit tumorigenicity in MDA-MB-231-xenograft models therefore suggesting that CCN5 is an anti-invasive molecule. The objective of the present study was to determine how CCN5 overexpression affects cell-cycle progression of breast cancer cells. We found knocking down of CCN5 in non-invasive breast cancer cells significantly downregulated the expression of p27Kip1, a tumor suppressor molecule. In contrast, enforced/ectopic expression of CCN5 overexpression in MDA-MB-231 breast cancer cells caused cell cycle arrest at G1 phase and dramatically reduced proliferation of these cells. CCN5 overexpression results in increased the stability as well as the expression of p27Kip1 protein. The regulation of p27Kip1 by CCN5 is mediated through AKT/FOXO3a signaling. Collectively, the studies suggest that CCN5 exerts its anti-invasive action through the regulation of p27Kip1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4207. doi:1538-7445.AM2012-4207

Aberrant expression of cell cycle regulatory genes predicts overall and disease free survival in malignant pleural mesothelioma patients

BackgroundMalignant pleural mesothelioma (MPM) is a highly aggressive disease with a generally poor prognosis. Since escape from cell cycle checkpoint control is common in several solid tumors, the present study was performed to evaluate the role of some cell cycle regulatory genes in the development and progression of MPM. Patients and methodsAberrant expression of p14ARF, p16INK4A, p21waf, p27KIP, p53, mdm2 and Rb was assessed in 55 MPM cases from Egypt using immunohistochemistry and PCR techniques. Results were correlated with clinico-pathological prognostic factors, overall and disease free survival (OS&DFS). ResultsAltered expression of p14ARF, p16INK4A, p21waf, p27KIP1, Rb, p53 and mdm2 proteins was detected in 50.9%, 54.5%, 53.3%, 61.8%, 53.3%, 58.2%, and 50.8% of cases, respectively. SV40 infection significantly correlated with p14ARF, 16INK4A, p27kip1 and Rb aberrations (p=0.014, p=0.02, p=0.01, p=−0.01). Asbestos exposure significantly correlated with p53, p21waf and mdm2 aberrations (p=0.001, p=0.03, p=0.02). On multivariate analysis PS≥2, p27KIP1 and Rb aberrations were independent prognostic factors for OS (p=0.016, p=0.011, p=0.003) whereas on tumor recurrence, p27KIP1 and Rb aberrations were independent prognostic factors for DFS (p=0.002, p=0.03, p=0.01). ConclusionsMPM is a complex disease characterized by multiple genetic aberrations; some of them involve cell cycle regulatory genes. p14ARF, p16INK4A, Rb and p27KIP1 seem to be involved in SV40-associated MPM whereas mdm2, p53 and p21WAF are related to asbestos exposure. In addition to recurrence and PS, only p27KIP1and Rb could be used as molecular prognostic markers in MPM.

Combinations of BRAF, MEK, and PI3K/mTOR Inhibitors Overcome Acquired Resistance to the BRAF Inhibitor GSK2118436 Dabrafenib, Mediated byNRASorMEKMutations

Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAF(V600E) and the YUSIT1 BRAF(V600K) melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1(K59del)) or NRAS mutation (NRAS(Q61K) and/or NRAS(A146T)) with and without MEK1(P387S) in the BRAF(V600E) background and NRAS(Q61K) in the BRAF(V600K) background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRAS(Q61K) or NRAS(A146T) in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1(K59del), but not MEK1(P387S), decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27(kip1) protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations.