P27 Kip1 Levels Research Articles

P27kip1 overexpression regulates IL-1β in the microenvironment of stem cells and eutopic endometriosis co-cultures

Endometriosis is a gynecological benign chronic disease defined as the growth of endometrial glands and stroma in extra-uterine sites, most commonly implanted over visceral and peritoneal surfaces within the female pelvis causing inflammatory lesions. It affects around 10% of the female population and is often accompanied by chronic pelvic pain, adhesion formation and infertility. Therefore, endometriosis could be considered a “social disease”, since it affects the quality of life, reproductivity and also has a socio-economic impact. The expression of cell cycle and inflammatory proteins is modified in the endometriotic tissues. Immunostaining of glandular and stromal cells in endometrial biopsies obtained from patients with endometriosis compared with those of healthy control demonstrated that endometriotic tissues have lower levels of p27kip1 protein. Endometriosis endometrial cells cultures have also lower levels of p27kip1 compared to health endometrial cells cultures and restore the cell cycle balance when transduced with an adenoviral vector carring the p27kip1 coding gene (Adp27EGFP). The low levels of p27kip1 are related to the S phase in the cell cycle, whereas higher levels lead to a G1 cell cycle arrest. The inflammatory cytokine IL-1β was recently identified as another key protein in the endometriosis proliferation. This cytokine has elevated levels during the proliferative and secretory phases of the menstrual cycle. In endometriosis endometrial cells cultures the IL-1β stimulates the production of IL-6 and IL-8, increasing the cell proliferation and reducing the apoptosis and Bax expression in these cells. According to these remarks, this work aims to evaluate the inflammatory effects in vitro, but more next to what happens in a woman’s body, associating endometrial cells with stem cells, thus mimicking the endometrial microenvironment, with gene therapy using Adp27, notoriously known as controller cell cycle, apoptosis and potent modulator of VEGF expression.

CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.

The DNA damage repair protein Ku70 regulates tumor cell and hepatic carcinogenesis by interacting with FOXO4

The capability for DNA double-strand breaks (DSBs) repair is crucial for chromatin dramatic changes and DNA damage in normal and tumor cells. We have investigated the clinicopathological significance of DNA repair gene Ku70 expression in hepatocellular carcinoma. We demonstrated that Ku70 expression was significantly increased in HCC, and the high expression levels were significantly correlated with gender, maximal tumor size, HBsAg status, tumor nodule number, distant metastasis and Ki-67 expression by clinicopathological analysis. The Kaplan–Meier survival curves revealed that increasing Ku70 expression was associated with poor prognosis in HCC patients. Ku70 expression was an independent prognostic marker of overall HCC patient survival in a multivariate analysis. In addition, through serum starvation and refeeding, we found that Ku70 was lowly expressed in serum-starved Huh7 and HepG2 HCC cells, and was progressively increased after serum-additioning. Furthermore, knockdown of Ku70 inhibited cell proliferation accompanying an increase in p27kip1 levels through interacting with FOXO4. These findings provide a rational framework for the progression of HCC and could be a potential molecular therapy by targeting the Ku70–FOXO4 interaction.

The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role.

Abstract A186: Low dose paclitaxel treatment increase the stability of p27Kip1

Abstract The purpose of our study is to better understand how taxanes (paclitaxel, docetaxel) induce cell death. Taxanes play a critical role in combination chemotherapy for many types of solid tumors, especially in advanced disease states. These drugs bind to tubulin in interphase and mitotic microtubules altering their dynamics. This leads to G2/M arrest that is thought to contribute to taxane-induced apoptosis, although the exact mechanisms remain elusive. We found that after treating MDA-MB-231 breast cancer cells with low dose (10X IC50 = 40 nM) paclitaxel over 4-48 h, the cyclin-dependent kinase inhibitor p27Kip1 progressively increases more than 2-fold. p27Kip1 is an important regulator of cell cycle, found in both the nucleus and cytoplasm, and its accumulation is associated with G1 arrest in most cells. Its tumor suppressor activity is associated with its nuclear localization, where it forms complexes with Cdk2 and cyclin E (or other cyclins) preventing DNA replication and cell cycle advance. In the cytoplasm, p27Kip1 was shown to enhance cell motility. When p27Kip1 becomes phosphorylated on Thr 187 leading to its ubiquitination and degradation, the cell cycle can progress. We pretreated cells with 10 μM cycloheximide for 1 h to prevent translational peptide elongation and found when cells were subsequently treated with 40 nM paclitaxel for up to 4 hours, quantitative Western blotting showed that p27Kip1 levels steadily increased, indicating that paclitaxel treatment stabilized p27Kip1 protein. Quantitative real-time PCR demonstrated a parallel increase in p27Kip1 mRNA over 8 hours and then a gradual decline in mRNA over 24-48 h, while the protein remained stable over this time period. Since paclitaxel binds to microtubules, we wanted to know whether association of p27Kip1 with microtubules might play a role in its stabilization. We isolated the intact microtubule cytoskeleton from cells using a 2M glycerol buffer (100 mM Tris pH 6.8, 0.5% Nonidet P-40, 1 mM MgCl2 and 2 mM EGTA) at 37°C and found that 20% of the total intracellular p27Kip1 is associated with the microtubule pellet in control cells and 40 nM paclitaxel treatment for 48 h reduces this amount by about half, demonstrating that paclitaxel treatment leads to an increased amount of p27Kip1 in the non-microtubule fraction of the cell lysate. Because the tumor suppressor function of p27Kip1 is associated with its nuclear localization, we isolated nuclear and cytoplasmic fractions from MDA-MB-231 cell lysates and found a 2-fold change in p27Kip1 with 40 nM paclitaxel treatment for 48 h, compared to cells treated with DMSO. The 2-fold increase in p27kip1 was found entirely in the cytoplasmic fraction. Western blotting with an antibody that recognizes p27Kip1 phosphorylated at Thr 187 surprisingly showed a nearly 2-fold increase in phospho- p27Kip1 with paclitaxel treatment in the cytoplasmic fractions. In summary, paclitaxel treatment of MDA-MB-231 breast cancer cells leads to a 2-fold increase in total p27Kip1. This increase is primarily in the cytoplasmic fraction and is associated with an increase in phospho-The 187-p27Kip1. Our data together suggest that paclitaxel treatment may block ubiquitination of phospho -Thr 187-p27Kip1 or its proteosomal degradation. These novel observations are important for understanding the regulation of p27Kip1 and the mode of action for paclitaxel. Citation Format: Sharon Lobert, Mary E. Graichen. Low dose paclitaxel treatment increase the stability of p27Kip1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A186.

Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity.

Although colorectal cancer (CRC) treatment with 5-fluorouracil (5-FU) is the first line of therapy for this debilitating disease, treatment effectiveness is often hampered by the development of drug resistance and toxicity at high doses. ER-β can play an important role in CRC development and possibly in its response to therapy. Pterostilbene (PT) possesses antioxidant and anticancer effects that are mediated by ER-β. In the current study, we test the hypothesis that PT sensitizes colon cancer cells to 5-FU and we examine the underlying mechanism(s) by which PT exerts its cytotoxic effects in CRC cells. Our data indicate that PT exhibited a more potent cytotoxic effect in Caco-2 compared to HCT-116 cells. PT/5-FU co-treatment was more effective in Caco-2 cells. Our data indicate that ER-β is expressed at higher levels in Caco-2 cells and its levels are further boosted with PT treatment. PT significantly suppressed Akt and ERK phosphorylations, and enhanced FOXO-1 and p27kip1 levels in Caco-2 cells. PT also induced a significant increase in Caco-2 cells at pre-G phase coupled with increased Bax/Bcl-2 ratio and PARP cleavage. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing ER-β protein.

Expression of Skp2 and p27kip1 proteins in hypopharyngeal squamous cell carcinoma and its clinical significance.

The aim of the present study was to determine the effect of S-phase kinase-associated protein 2 (Skp2) and cyclin-dependent kinase inhibitor p27kinase-interacting protein 1 (p27kip1) protein expression on the occurrence and development of hypopharyngeal squamous cell carcinoma. The protein expression levels of Skp2 and p27kip1 were detected in 42 hypopharyngeal squamous cell carcinoma and 15 normal hypopharyngeal mucous membrane specimens using the an immunohistochemical PV-9000 two-step method. The expression levels of Skp2 protein were significantly different in hypopharyngeal squamous cell carcinomas and normal hypopharyngeal mucous membranes (61.90 vs. 26.67%; P<0.05). By contrast, the protein expression levels of Skp2 were significantly positively correlated with tumor T stage (rs=0.329, P<0.05) and cervical lymph node metastasis (rs=0.402, P<0.05). Furthermore, the expression levels of p27kip1 protein were significantly different in hypopharyngeal squamous cell carcinomas and normal hypopharyngeal mucous membranes (11.9 vs. 53.33%; P<0.05), while p27kip1 protein expression was significantly negatively correlated with tumor T-stage (rs=-0.351, P<0.05) and cervical lymph node metastasis (rs=-0.371, P<0.05). Notably, a significant negative correlation was observed between the expression levels of Skp2 and p27kip1 proteins in hypopharyngeal squamous cell carcinoma (P<0.05). In addition, abnormal expression levels of Skp2 and p27kip1 proteins were observed in hypopharyngeal squamous cell carcinoma tissues. Thus, Skp2 and p27kip1 proteins may be involved in the development of hypopharyngeal squamous cell carcinoma. The current study proposed that combined detection of Skp2 and p27kip1 may be useful for assessing the characteristics and prognosis of hypopharyngeal squamous cell carcinoma.

Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D.

The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 μmol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin that controls cell-cycle progression.

Differential somatostatin receptor (SSTR) 1–5 expression and downstream effectors in histologic subtypes of growth hormone pituitary tumors

PurposeThe aim of this study was to examine whether differential expression of somatostatin receptors (SSTR) 1–5 and downstream effectors are different in densely (DG) and sparsely (SG) granulated histological growth hormone (GH) pituitary tumor subtypes. MethodsThe study included 33 acromegalic patients with 23 DG and 10 SG tumors. SSTR1–5 were measured by qPCR and immunoblotting. Signaling candidates downstream of SSTR2 were also assessed. ResultsSSTR2 mRNA and protein levels were significantly higher in DG compared to SG tumors. Downstream of SSTR2, p27kip1 was decreased (2.6-fold) in SG compared to DG tumors, suggesting a potential mechanism of SSA resistance in SG tumors with intact SSTR2 expression. Re-expression of E-cadherin in GH pituitary cell increased p27kip1 levels. ConclusionsHistological subtyping correlated with SSTR2, E cadherin and p27kip protein levels and these may serve as useful biomarkers in GH tumors to predict behavior and response to therapy with SSA.

LIM domain only 2 induces glioma invasion via cytosolic p27(KIP1).

High-grade gliomas are considered the most malignant of brain tumors and have a poor prognosis. In a previous study, we showed that LIM domain only 2 (LMO2) regulates glioma stem cell properties and tumor angiogenesis and gave rise to highly invasive glioma xenografts. Glioma invasion in the surrounding parenchymal tissues is a major hurdle with respect to eliminating glioma by surgery. Invasive glioma cells are considered one of the main culprits for the recurrence of tumors after therapies. In the current study, we focused on determining the molecular mechanism(s) by which LMO2 regulates glioma cell migration and invasion. Forced expression of LMO2 in human U87MG glioma cells led to glioma invasion, as determined by in vivo xenograft assays and enhanced in vitro migration and invasion. LMO2 was associated with increased levels of cytosolic p27(Kip1) protein. LMO2 possibly induced the stabilization and augmented interactions between p27(Kip1) and RhoA. We knocked down the expression of p27(Kip1), which led to a decrease in LMO2-driven glioma cell migration and invasion. Taken together, our findings indicate that LMO2 promotes glioma cell migration and invasion by increasing the levels of cytosolic p27(Kip1).

Inhibition of Notch3 prevents monocrotaline-induced pulmonary arterial hypertension

ABSTRACTIt has been shown that activation of Notch3 signaling is involved in the development of pulmonary arterial hypertension (PAH) by stimulating pulmonary arteries remodeling, while the molecular mechanisms underlying this are still largely unknown. The aims of this study are to address these issues. Monocrotaline dramatically increased right ventricle systolic pressure to 39.0 ± 2.6 mmHg and right ventricle hypertrophy index to 53.4 ± 5.3% (P < 0.05 versus control) in rats, these were accompanied with significantly increased proliferation and reduced apoptosis of pulmonary vascular cells as well as pulmonary arteries remodeling. Treatment of PAH model with specific Notch inhibitor DAPT significantly reduced right ventricle systolic pressure to 26.6 ± 1.3 mmHg and right ventricle hypertrophy index to 33.5 ± 2.6% (P < 0.05 versus PAH), suppressed proliferation and enhanced apoptosis of pulmonary vascular cells as well as inhibited pulmonary arteries remodeling. Our results further indicated that level of Notch3 protein and NICD3 were increased in MCT-induced model of PAH, this was accompanied with elevation of Skp2 and Hes1 protein level and reduction of P27Kip1. Administration of rats with DAPT-prevented MCT induced these changes. Our results suggest that Notch3 signaling activation stimulated pulmonary vascular cells proliferation by Skp2-and Hes1-mediated P27Kip1 reduction, and Notch3 might be a new target to treat PAH.

Digoxin inhibits PDGF-BB-induced VSMC proliferation and migration through an increase in ILK signaling and attenuates neointima formation following carotid injury

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) are key events in the development of artery restenosis following percutaneous coronary intervention. Digoxin has long been used in the treatment of heart failure and has been shown to inhibit the proliferation of cancer cells through multiple pathways. However, the potential role of digoxin in the regulation of VSMC proliferation and migration and its effectiveness in the treatment of cardiovascular diseases, such as restenosis, remains unexplored. In the present study, we demonstrate that digoxin-induced growth inhibition is associated with the downregulation of CDK activation and the restoration of p27Kip1 levels in platelet-derived growth factor (PDGF)-stimulated VSMCs. In addition, we found that digoxin restored the PDGF-BB-induced inhibition of integrin linked kinase (ILK) expression and prevented the PDGF-BB-induced activation of glycogen synthase kinase (GSK)-3β. Furthermore, digoxin inhibited adhesion molecule and extracellular matrix relative protein expression. Finally, we found that digoxin significantly inhibited neointima formation, accompanied by a decrease in cell proliferation following vascular injury in rats. These effects of digoxin were shown to be mediated, at least in part, through an increase in ILK/Akt signaling and a decrease in GSK-3β signaling in PDGF-BB-stimulated VSMCs. In conclusion, our data demonstrate that digoxin exerts an inhibitory effect on the PDGF-BB-induced proliferation, migration and phenotypic modulation of VSMCs, and prevents neointima formation in rats. These observations indicate the potential therapeutic application of digoxin in the treatment of cardiovascular diseases, such as restenosis.

Abstract 4468: Anticancer activity of novel cucurbitacin analogue in pancreatic cancer

Abstract Background: Human pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States. Accumulating studies have witnessed the malfunction of many chemotherapeutic regimens and the current standard-of-care therapy, gemcitabine (GEM), enhances patient survival by only few months. Cucurbitacins, naturally occurring dietary tetracyclic triterpenoid compounds, have shown promising anti-cancer activities. Herein, we investigated the potency and anti-cancer efficacy of a novel analogue of cucurbitacin (Cuc D) in pancreatic cancer cell lines and in a xenograft mouse model. Additionally, we determined efficacy of this analogue on MicroRNAs (miRNAs) which are important regulators of genes that have crucial roles in pancreatic tumorigenesis. Methods: The effect of Cuc D on the growth of pancreatic cancer cells was determined by cell proliferation assay using six pancreatic cancer (MiaPaCa-2, CaPan-1, HPAF-II, Panc-1, BxPC-3 and AsPc-1) cells. Cell growth kinetic assay was carried out at 24, 48, 72 and 96 h. The clonogenic potential of cancer cells was also studied using the colony formation assay. Tumor suppressor miR-145, which is downregulated in pancreatic cancer, directly target MUC13. Thus the effect of Cuc D was also investigated on the expression of miR-145 through qPCR analysis. Immunoblotting techniques were performed to study the known direct targets of miRNA-145 and its associated proteins. The anti-cancer potential of Cuc D in pancreatic cancer was also evaluated in vivo using a xenograft mouse model. Results: Our results demonstrate potent anticancer effects of Cuc D on pancreatic cancer cells. Cuc D induces dose and time dependent inhibition of cell proliferation in a panel of gemcitabine sensitive/resistant pancreatic cancer cell line models at nanomolar concentrations (100-500 nM). It also inhibits colony formation and invasiveness of pancreatic cancer cells. Furthermore, Cuc D blocks the cell cycle progression in G2/M phase and decreases the mitochondrial membrane potential in pancreatic cancer cells. Notably, Cuc D significantly increases the expression of tumor suppressor miR-145 in HPAF-II cells as observed by qPCR. Furthermore, Cuc D decreases the expression of MUC13 and its associated proteins including pAKT and HER2. In addition, it restores the expression of p53 level as studied by immunofluorescence technique. The expression of key oncogenic proteins including NF-κB, STAT3 (Tyr-705) and Mcl-1 were also downregulated. The levels of PTEN and p27 (Kip1) tumor suppressor genes were increased after Cuc D treatment. Additionally, in vivo administration of Cuc D effectively inhibited pancreatic tumor growth in xenograft mouse model. Conclusion: Overall, this study suggests that Cuc D modulates the expression of key oncogenes and tumor suppressors, thus it can be a promising therapeutic modality for pancreatic cancer prevention and treatment. Citation Format: Mohammed Sikander, Sheema Khan, Neeraj Chauhan, Mohd Saif Zaman, Murali Mohan Yallapu, Fathi T. Halaweish, Bhavin Chauhan, Shabnam Malik, Meena Jaggi, Subhash C. Chauhan. Anticancer activity of novel cucurbitacin analogue in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2015-4468

Abstract 2036: Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth

Abstract One of the most fundamental traits of cancer cells is the ability to sustain proliferation by circumventing cell cycle regulatory programs normally controlled by tumor suppressors. Keratin 17 (K17) is not expressed in most somatic tissues but is overexpressed and is a negative prognostic marker in cervical, breast, ovarian, and gastric carcinomas. The mechanisms by which K17 contributes to cancer-related signaling, however, remain unknown. Here, we report that nuclear-localized K17 interacts with tumor suppressor p27KIP1 and mediates its nuclear export, maintaining proliferation. After validating K17 is a prognostic marker in cervical cancer, independent of tumor stage, we performed in vitro and in vivo experiments to investigate the role of K17 in proliferative signaling and tumor growth using loss- and gain- of function approaches in cervical cancer and other cancer-derived cell lines. We found that K17 expression maintains proliferation, while silencing K17 induces G1 arrest by nuclear accumulation and stabilization of tumor suppressors p27KIP1 and pRb. During G1, K17 localizes in the nucleus, mediated by a classical bipartite nuclear localization signal (NLS) (AA385-400) that was identified by in silico analysis. This NLS is specific among keratins and present only in primates but not in other species. To our knowledge, this is the first report that a keratin has a NLS and is capable of undergoing nuclear translocation. During G1, p27KIP1is exported from the nucleus in a CRM1-dependent manner and is subsequently degraded, triggering G1/S transition. p27KIP1 lacks the classical leucine-rich nuclear export signal (NES) and requires an adaptor for CRM1-exportin binding. After confirming the binding of K17 and p27KIP1 within the nucleus, we performed in silico analyses and identified a leucine-rich NES required for CRM1-binding in K17 (AA194-199), which was further validated by a 3-fold nuclear retention of K17 and p27KIP1 after leptomycin B treatment. We introduced point mutations within the K17-NES (mNES) and the K17-NLS (mNLS). Nuclear p27KIP1 was lost in cells expressing wild-type K17. In contrast, nuclear p27KIP1 levels were 3-fold higher in cells expressing either mNLS or mNES. Furthermore, nuclear localization of K17 was abolished in mNLS cells. Xenografts of cervical cancer cells showed that tumors derived from K17 expressing cells were 3-times larger than those derived from K17 knockdown cells, the latter showing increased nuclear p27KIP1 and decreased PCNA and Ki67 expression. Finally, we identified an inverse correlation between K17 and p27KIP1 expression in human cervical cancer specimens, intertumorally and intratumorally. These data suggest a model in which nuclear-localized K17 acts as an oncoprotein promoting G1/S transition in cancer cells, by mediating exportin binding and nuclear translocation of tumor suppressor p27KIP1, contributing to sustained proliferation, tumor aggressiveness and poor-patient outcome. Citation Format: Luisa F. Escobar-Hoyos, Ruchi Shah, Lucia Roa-Peña, Nilofar Najafian, Elizabeth Vanner, Anna Banach, Erik Nielsen, Ramsey Al-Khalil, Ali Akalin, David Talmage, Kenneth Shroyer. Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2015-2036

Treatment with cucurbitacin B alone and in combination with gefitinib induces cell cycle inhibition and apoptosis via EGFR and JAK/STAT pathway in human colorectal cancer cell lines.

The epidermal growth factor receptor (EGFR) associated with signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), plays an important role in colorectal cancers (CRCs). Gefitinib (Gef) is an orally active inhibitor targeting the adenosine tri phosphate-binding domain of EGFR, and cucurbitacin B (CuB) is a selective inhibitor of JAK/STAT signaling with potent antitumor activity via suppression of STAT3 phosphorylation, but the underlying mechanism is not clear. We aimed to investigate the apoptotic and antiproliferative effects of CuB as a single agent and in combination with Gef on both HT-29 and HCT-116 cell lines. Cell proliferation, cell cycle distribution, and apoptosis were evaluated using viability assay, fluorescent microscopy, cytotoxicity assay, proliferation, DNA fragmentation, and cleaved caspase 3 levels. Real-time polymerase chain reaction and Western blot analyses were performed to determine the expression of relevant genes and proteins including antiapoptotic, proapoptotic, and cell cycle regulation. EGFR, phosphorylated EGFR (pEGFR), STAT3, and pSTAT3 proteins were evalutaed with Western blot analysis. Our results showed that, compared to CuB alone, CuB plus Gef treatment caused a significant growth and cell cycle inhibition and induced apoptosis in both cell lines. Also CuB plus Gef treatment decreased DNA synthesis rate more effectively than CuB alone. Treatment with CuB alone and in combination with Gef decreased the expression levels of B-Cell CLL/Lymphoma 2 (Bcl-2), BCL2-like 1 (BCL2L1), cyclin D1, pSTAT3, and pEGFR and increased the expression levels of Bcl-2-like protein 4, Bcl-2 homologous antagonist/killer, Bcl-2-associated death promoter, Bcl-2-like protein 11, and p27kip1 levels. Our results suggest that treatment with CuB alone and more likely in combination with Gef may be a considerable alternative therapeutic approach for CRC, at least in vitro.

The comparison between dual inhibition of mTOR with MAPK and PI3K signaling pathways in KRAS mutant NSCLC cell lines.

KRAS mutations are found in 15-25 % of patients with lung adenocarcinoma, and they lead to constitutive activation of KRAS signaling pathway that results in sustained cell proliferation. Currently, there are no direct anti-KRAS therapies available. Therefore, it is rational to target the downstream molecules of KRAS signaling pathway, which are mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK) and PI3K pathway (PI3K-AKT-mTOR). Here, we examined the inhibition of both these pathways alone and in combination and analyzed the anti-proliferative and apoptotic events in KRAS mutant NSCLC cell lines, A549 and Calu-1. Cytotoxicity was determined by MTT assay after the cells were treated with LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), and RAD001 (mTOR inhibitor) for 24 and 48 h. The expression levels of p-ERK, ERK, AKT, p-AKT, p53, cyclinD1, c-myc, p27(kip1), BAX, BIM, and GAPDH were detected by western blot after 6 and 24 h treatment. Although PI3K/mTOR inhibition is more effective in cytotoxicity in A549 and Calu-1 cells, MEK/mTOR inhibition markedly decreases cell proliferation protein marker expressions. Our data show that combined targeting of MEK and PI3K-AKT with mTOR is a better option than single agents alone for KRAS mutant NSCLC, thus opening the possibility of a beneficial treatment strategy in the future.

A Basal Tone of 2-Arachidonoylglycerol Contributes to Early Oligodendrocyte Progenitor Proliferation by Activating Phosphatidylinositol 3-Kinase (PI3K)/AKT and the Mammalian Target of Rapamycin (MTOR) Pathways

A basal tone of the endocannabinoid 2-arachidonoylglycerol (2-AG) enhances late oligodendrocyte progenitor cell (OPC) differentiation. Here, we investigated whether endogenous 2-AG may also promote OPC proliferation in earlier stages. We found that the blockade of 2-AG synthesizing enzymes, sn-1-diacylglycerol lipases α and β (DAGLs), with RHC-80267 or the antagonism of either CB1 or CB2 cannabinoid receptors with AM281 and AM630, respectively, impaired early OPC proliferation stimulated by platelet-derived growth factor (PDGF-AA) and basic fibroblast growth factor (bFGF). On the contrary, increasing the levels of endogenous 2-AG by blocking the degradative enzyme monoacylglycerol lipase (MAGL) with JZL-184, significantly increased OPC proliferation as did agonists of cannabinoid receptor CB1 (ACEA), CB2 (JWH133) or both (HU-210). To elucidate signaling pathways underlying OPC proliferation, we studied the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt and its downstream target mammalian target of rapamycin (mTOR). We show that phosphorylation of Akt and mTOR is required for OPC proliferation stimulated by growth factors (PDGF-AA and bFGF) or by CB1/CB2 agonists (ACEA/JWH133), since it was strongly decreased after LY294002 or rapamycin treatment. In line with this, blockade of CB1 (AM281), CB2 (AM630) or DAGLs (RHC-80267), decreased phosphorylation of Akt, mTOR and 4E-BP1, diminished cyclin E-cdk2 complex association and increased p27(kip1) levels. Our data suggest that proliferation of early OPCs stimulated by PDGF-AA and bFGF depends on the tonic activation of cannabinoid receptors by endogenous 2-AG and provide further evidence on the role of endocannabinoids in oligodendrocyte development, being important for the maintenance and self-renewal of the OPCs. The results highlight the therapeutic potential of the endocannabinoid signaling in the emerging field of brain repair.

COP1 enhances ubiquitin-mediated degradation of p27Kip1 to promote cancer cell growth.

p27 is a critical CDK inhibitor involved in cell cycle regulation, and its stability is critical for cell proliferation. Constitutive photomorphogenic 1 (COP1) is a RING-containing E3 ubiquitin ligase involved in regulating important target proteins for cell growth, but its biological activity in cell cycle progression is not well characterized. Here, we report that p27Kip1 levels are accumulated in G1 phase, with concurrent reduction of COP1 levels. Mechanistic studies show that COP1 directly interacts with p27 through a VP motif on p27 and functions as an E3 ligase of p27 to accelerate the ubiquitin-mediated degradation of p27. Also, COP1-p27 axis deregulation is involved in tumorigenesis. These findings define a new mechanism for posttranslational regulation of p27 and provide insight into the characteristics of COP1-overexpressing cancers.

Effects of dihydrotestosterone on rat dermal papilla cells in vitro

Androgenetic alopecia involves the action of dihydrotestosterone (DHT) on dermal papilla cells (DPCs) that line the base of the hair follicle. However, the mechanism of DHT action is not completely understood. The effects of DHT on DPCs, regulatory cells that function in follicle growth and the hair cycle, were examined in immortalized cells derived from rat vibrissa follicles. DHT did not affect the proliferation of immortalized DPCs. However, flow cytometry analysis revealed that DHT increased cell-cycle arrest in these cells, which was accompanied by an increase in the p27kip1 level and by decreases in cyclin E, cyclin D1, and cyclin-dependent kinase 2 levels. DHT treatment resulted in the phosphorylation and nuclear translocation of Smad2/3, a mediator of the transforming growth factor-β (TGF-β) signaling pathway, which leads to the catagen phase of the hair cycle. DHT also induced the phosphorylation and nuclear translocation of heat shock protein 27 (HSP27). Moreover, DHT decreased the levels of total and nuclear β-catenin, an important regulator of hair growth and proliferation, while lithium chloride, a glycogen synthase kinase-3β inhibitor, attenuated the DHT-induced downregulation of the β-catenin level. On the other hand, DHT increased the phosphorylation of mammalian target of rapamycin (mTOR), a regulator of proliferation, in immortalized DPCs. These results illustrate that DHT could shorten the duration of the hair growth cycle by initiating cell-cycle arrest, downregulating the β-catenin level, and upregulating the TGF-β/Smad and HSP27 level, whereas activation of mTOR by DHT could attenuate the inhibition of hair growth cycle in immortalized DPCs.

Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis.

Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non‐permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27Kip1 were observed in cathepsin B‐deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27Kip1 within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy. © 2015 The Authors. Molecular Carcinogenesis, published by Wiley Periodicals, Inc.