Cyclin-dependent Kinase Inhibitor P27 Kip1 Research Articles

Canagliflozin Mediates Effective Blockade of the mTORC1 - HIF-1a Pathway, Inhibits Growth and Enhances Non-Small Cell Lung Cancer (NSCLC) Response to Radiotherapy (RT)

Alterations in NSCLC cell metabolism contribute to rapid tumor growth, resistance to RT and poor prognosis. The mammalian Target of Rapamycin Complex 1 (mTORC1) - Hypoxia-Inducible Factor-1α (HIF-1α) pathway mediates expression of the Warburg glycolytic phenotype in NSCLC and induces RT resistance. Earlier, we showed that the new diabetes agent Canagliflozin (CANA), developed to control hyperglycemia through inhibition of the renal Na+-glucose co-transporter 2 (SGLT2), also inhibits mitochondrial complex I respiration and blocks mTORC1 signaling. In this study, we analyzed CANA’s transcriptional and post-translational effects and its regulation on RT response in preclinical models of NSCLC. Cell proliferation and clonogenic survival assays were used to assess the efficacy of CANA (0–30 μM) and RT (0–8 Gy) in adenocarcinoma (A549, H1299, H1975) and squamous cell carcinoma (SK-MES-1) NSCLC cells. Protein immunoblotting, RT-qPCR and RNAseq of A549 NSCLC cells were performed to study regulation of pathway activation and gene expression in response to CANA and RT. In-vivo effects of the two agents were examined with ectopic A549 xenografts grown in immunodeficient nude mice treated with CANA (100 mg/kg/day in chow diet) and/or RT (5 Gy). RNAseq showed that clinically achievable CANA doses (10 μM) mediate profound regulation of NSCLC cell transcriptional activity with over 100 upregulated and 80 downregulated genes. CANA significantly downregulates HIF-1α pathway-related genes that mediate HIF-1α stability and nuclear transfer, glycolysis and lipogenesis, tyrosine kinase receptor signaling and inhibition of apoptosis. CANA treatment led to activation of AMP-activated protein kinase (AMPK) and blockade of the mammalian target of rapamycin complex 1 (mTORC1) pathway. It abolished phosphorylation of the mitosis marker, histone H3. This was consistent with long-term induction of the G1/S cyclin-dependent kinase inhibitor p27kip1. Clinically relevant doses of CANA suppressed proliferation and clonogenic survival in all NSCLC cell lines but this was enhanced in the K-Ras mutant/LKB1null A549 cells. Combination index analysis indicated additivity and sensitization interaction between the CANA and RT anti-proliferative effects. In the A549 xenograft model, CANA enhanced the cytotoxic effects of RT in an additive manner. CANA mediates effective suppression of the mTORC1-HIF-1α pathway, regulates gene expression and mediates potent anti-tumor activity in NSCLC models in-vitro and in-vivo. Given that CANA is a widely-used, well-tolerated and economical agent, this work indicates strong potential for this agent to be investigated rapidly in NSCLC clinical trials in combination with RT.

RFWD2 induces cellular proliferation and selective proteasome inhibitor resistance by mediating P27 ubiquitination in multiple myeloma

RFWD2 induces cellular proliferation and selective proteasome inhibitor resistance by mediating P27 ubiquitination in multiple myeloma

P27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth.

Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27-/- cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27-/- cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.

Corrigendum.

Development, Growth & DifferentiationVolume 62, Issue 6 p. 462-462 CORRIGENDUMFree Access Corrigendum This article corrects the following: Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming Asuka Takehara, Yasuhisa Matsui, Volume 61Issue 6Development, Growth & Differentiation pages: 357-364 First Published online: June 14, 2019 First published: 26 August 2020 https://doi.org/10.1111/dgd.12672AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Takehara and Mtasui (2019), the authors note that, in the abstract, a sentence ‘We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1-S transition and is transcriptionally repressed by p53, was significantly downregulated by AKT activation.’ should be ‘We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1-S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation.’ The authors would like to apologize for any inconvenience caused. REFERENCE Takehara, A, & Mtasui, Y (2019). Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming. Development Growth and Differentiation, 61(6), 357– 364. https://doi.org/10.1111/dgd.12621 Volume62, Issue6August 2020Pages 462-462 ReferencesRelatedInformation

Modulatory Effect of Indoles on the Expression of miRNAs Regulating G1/S Cell Cycle Phase in Breast Cancer Cells.

Indole-3-carbinol (I3C) is a naturally occurring glucosinolate found in Brassica vegetables that is usually converted in gastric acidic environment to the efficient metabolite 3,3'-diindolylmethane (DIM). Both indoles (I3C and DIM) are known chemopreventive agents for various cancers including breast cancer. This study aimed to investigate the influence of both indoles on the tumor suppressor miRNAs (let-7a-e, miR-15a, miR-16, miR-17-5p, miR-19a, and miR-20a) and oncomiRs (miR-181a, miR-181b, miR-210, miR-221, and miR-106a), which are controlling the cell cycle key regulators: cyclin-dependent kinases (CDKs), CDK inhibitor p27Kip1, and cyclin D1. Our results indicated that both indoles generally elevated the expression of the tumor suppressor miRNAs let-7a-e, miR-19a, miR-17-5p, and miR-20a and decreased the expression of the oncomiR list. Both indoles were able to significantly suppress the expression of CDK4 and CDK6 as well as the apoptotic markers Bcl-2 and survivin. Both indoles decreased cyclin-D1 protein, where I3C decreased cytoplasmic and nuclear cyclin-D1 significantly. Cytoplasmic and nuclear P27Kip1 showed overexpression following treatment with I3C higher than that detected following DIM treatment. This study provides a mechanistic elucidation of the previously reported cell cycle arrest by I3C and DIM in breast cancer cells suggesting that this effect could be through modulation of miRNAs expression that, in turn, regulates the genetic network controlling the G1/S phase in cell cycle progression.

Abstract A74: The HIV protease inhibitor nelfinavir, alone or in combination with the proteasome inhibitor bortezomib, is cytotoxic to high-grade serous ovarian cancer cells regardless of platinum sensitivity

Abstract High-grade serous ovarian cancer (HGSOC) is a deadly disease, affecting women worldwide. Major challenges in treating HGSOC patients involve overcoming resistance to platinum (Pt)-based therapy by developing novel treatment approaches. Nelfinavir (NFV), an anti-HIV drug, has shown to be effective against cancers of high secretory capacity—e.g., multiple myeloma—by aggravating the stress of the endoplasmic reticulum (ER). Its potential therapeutic role against HGSOC, however, remains unclear. We hypothesized that simultaneous ER stress aggravation by NFV and blockage of the proteolytic capacity of the proteasome with a proteasome inhibitor should cause sufficient ER stress to tilt cells to a death fate. In this study, we investigated the cytotoxicity of the ER aggravator NFV as monotherapy and in combination with the proteasome inhibitor bortezomib (BZ) against HGSOC cells having differential sensitivities to cisplatin (CDDP). NFV caused cell cycle arrest associated with increased expression of cyclin-dependent kinase inhibitor p27kip1, while triggering the unfolded protein response (UPR) in a dose-dependent manner and assessed by increased subrogate ER stress biomarkers GRP78 and CHOP. We also discovered that blocking the ubiquitin proteasome system (UPS) using cytostatic concentrations of BZ causes accumulation of poly-ubiquitinated proteins and further activation of the UPR. More importantly, when we combined BZ with NFV, we observed a potentiation of the action of BZ leading to HGSOC lethality regardless of Pt sensitivity. These results suggest that NFV could potentially be used following standard Pt-based treatment, either as monotherapy or in conjunction with proteasome inhibitors, to kill the remaining HGSOC cells that otherwise recur and recreate the disease. In addition, the data provide the rationale for rapid repurposing to treat HGSOC of two drugs clinically approved for other uses, as their systemic toxicities have already been assessed. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Michael A. Lisio, Carlos M. Telleria. The HIV protease inhibitor nelfinavir, alone or in combination with the proteasome inhibitor bortezomib, is cytotoxic to high-grade serous ovarian cancer cells regardless of platinum sensitivity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A74.

Role of the imprinted allele of the Cdkn1c gene in mouse neocortical development

Imprinted genes are expressed from only one allele in a parent of origin–specific manner. The cyclin-dependent kinase inhibitor p57kip2 is encoded by an imprinted gene Cdkn1c, with the paternal allele being silenced. The possible expression and function of the paternal allele of Cdkn1c have remained little studied, however. We now show that the paternal allele of the Cdkn1c gene is expressed at a low level in the developing mouse neocortex. Surprisingly, the central nervous system-specific conditional deletion of the paternal allele (pat cKO) at the Cdkn1c locus resulted in a marked reduction in brain size. Furthermore, pat cKO gradually reduced the number of neural stem-progenitor cells (NPCs) during neocortical development, and thus reduced the number of upper-layer neurons, which were derived from late-stage NPCs. Our results thus show that the paternal allele of the Cdkn1c locus plays a key role in maintenance of NPCs during neocortical development.

WIP1 dephosphorylation of p27Kip1 Serine 140 destabilizes p27Kip1 and reverses anti-proliferative effects of ATM phosphorylation

ABSTRACT The phosphoinositide-3-kinase like kinases (PIKK) such as ATM and ATR play a key role in initiating the cellular DNA damage response (DDR). One key ATM target is the cyclin-dependent kinase inhibitor p27Kip1 that promotes G1 arrest. ATM activates p27Kip1-induced arrest in part through phosphorylation of p27Kip1 at Serine 140. Here we show that this site is dephosphorylated by the type 2C serine/threonine phosphatase, WIP1 (Wildtype p53-Induced Phosphatase-1), encoded by the PPM1D gene. WIP1 has been shown to dephosphorylate numerous ATM target sites in DDR proteins, and its overexpression and/or mutation has often been associated with oncogenesis. We demonstrate that wildtype, but not phosphatase-dead WIP1, efficiently dephosphorylates p27Kip1 Ser140 both in vitro and in cells and that this dephosphorylation is sensitive to the WIP1-specific inhibitor GSK 2830371. Increased expression of wildtype WIP1 reduces stability of p27Kip1 while increased expression of similar amounts of phosphatase-dead WIP1 has no effect on p27Kip1 protein stability. Overexpression of wildtype p27Kip1 reduces cell proliferation and colony forming capability relative to the S140A (constitutively non-phosphorylated) form of p27. Thus, WIP1 plays a significant role in homeostatic modulation of p27Kip1 activity following activation by ATM.

Imprinted Cdkn1c genomic locus cell-autonomously promotes cell survival in cerebral cortex development

The cyclin-dependent kinase inhibitor p57KIP2 is encoded by the imprinted Cdkn1c locus, exhibits maternal expression, and is essential for cerebral cortex development. How Cdkn1c regulates corticogenesis is however not clear. To this end we employ Mosaic Analysis with Double Markers (MADM) technology to genetically dissect Cdkn1c gene function in corticogenesis at single cell resolution. We find that the previously described growth-inhibitory Cdkn1c function is a non-cell-autonomous one, acting on the whole organism. In contrast we reveal a growth-promoting cell-autonomous Cdkn1c function which at the mechanistic level mediates radial glial progenitor cell and nascent projection neuron survival. Strikingly, the growth-promoting function of Cdkn1c is highly dosage sensitive but not subject to genomic imprinting. Collectively, our results suggest that the Cdkn1c locus regulates cortical development through distinct cell-autonomous and non-cell-autonomous mechanisms. More generally, our study highlights the importance to probe the relative contributions of cell intrinsic gene function and tissue-wide mechanisms to the overall phenotype.

Loss of p27Kip1 leads to expansion of CD4+ effector memory T cells and accelerates colitis-associated colon cancer in mice with a T cell lineage restricted deletion of Smad4

ABSTRACT The cyclin-dependent kinase inhibitor p27Kip1 is a tumor suppressor whose intrinsic activity in cancer cells correlates with tumor aggressiveness, invasiveness, and impaired tumor cell differentiation. Here we explore whether p27Kip1 indirectly influences tumor progression by restricting expansion and survival of effector memory T cell (TEM) populations in a preclinical model of spontaneous colitis-associated colorectal cancer (CAC). We show mRNA and protein expression of p27Kip1 to be significantly decreased in the colons of mice with a T cell-restricted deletion of the TGF-β intermediate, SMAD4 (Smad4TKO). Loss of p27Kip1 expression in T cells correlates with the onset of spontaneous CAC in Smad4TKO mice by 8 months of age. This phenotype is greatly accelerated by the introduction of a germline deletion of CDKN1b (the gene encoding p27Kip1) in Smad4TKO mice (Smad4TKO/p27Kip1-/-, DKO). DKO mice display colon carcinoma by 3 months of age and increased mortality compared to Smad4TKO. Importantly, the phenotype in DKO mice is associated with a significant increase in the frequency of effector CD4 T cells expressing abundant IFN-γ and with a concomitant decrease in Foxp3+ regulatory T cells, both in the intestinal mucosa and in the periphery. In addition, induction of inflammatory mediators (IFN-γ, TNF-γ, IL-6, IL-1β, iNOS) and activation of Stat1, Stat3, and IκB is also observed in the colon as early as 1–2 months of age. Our data suggest that genomic alterations known to influence p27Kip1 abundance in gastrointestinal cancers may indirectly promote epithelial malignancy by augmenting the production of inflammatory mediators from a spontaneously expanding pool of TEM cells.

Antitumor effect of baicalin from the Scutellaria baicalensis radix extract in B-acute lymphoblastic leukemia with different chromosomal rearrangements

Acute B-lymphoblastic leukemia (B-ALL) is the most common hematologic malignancy in children. Many cases of B-ALL harbor chromosomal translocations which are often critical determinants of prognosis. Most of them represent altered transcription factors that impact gene transcription or enhance signaling. B-ALLs harboring the mixed-lineage leukemia 1 (MLL1) gene rearrangements represent aggressive, high-risk type of early childhood leukemias that are usually associated with a very poor prognosis. Therefore, there is an urgent need for novel therapeutic agents as well as new treatment strategies.The objective was to examine the vitro inhibitory effects of Scutellaria baicalensis root extract (SBE) in B-ALL cell lines with different chromosomal rearrangements and in leukemic blasts derived from patients’ bone marrow (BMCs).In this study we showed that baicalin which is the main component of the SBE possess antitumor activity against all leukemic cell lines especially those with MLL and PBX1 gene rearrangements. Baicalin inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase, and induced cell death through caspase 3/7 activation. Moreover, baicalin treatment inhibited the glycogen synthase kinase-3β (GSK‐3β) by suppressing its phosphorylation at Y216, and upregulated the downstream mediator of the cell cycle arrest - cyclin dependent kinase inhibitor p27Kip1. Bone marrow derived blasts from B-ALL patients also exhibited varied sensitivity towards baicalin with 72% patients sensitive to the SBE and baicalin treatment.Taken together, our findings provide new insights into the anti-cancer properties of baicalin by showing its diverse mode of action which might be related to the different genetic background.

Pharmacological Stimulation of Nurr1 Promotes Cell Cycle Progression in Adult Hippocampal Neural Stem Cells.

Nuclear receptor related-1 (Nurr1) protein performs a crucial role in hippocampal neural stem cell (hNSC) development as well as cognitive functions. We previously demonstrated that the pharmacological stimulation of Nurr1 by amodiaquine (AQ) promotes spatial memory by enhancing adult hippocampal neurogenesis. However, the role of Nurr1 in the cell cycle regulation of the adult hippocampus has not been investigated. This study aimed to examine changes in the cell cycle-related molecules involved in adult hippocampal neurogenesis induced by Nurr1 pharmacological stimulation. Fluorescence-activated cell sorting (FACS) analysis showed that AQ improved the progression of cell cycle from G0/G1 to S phase in a dose-dependent manner, and MEK1 or PI3K inhibitors attenuated this progression. In addition, AQ treatment increased the expression of cell proliferation markers MCM5 and PCNA, and transcription factor E2F1. Furthermore, pharmacological stimulation of Nurr1 by AQ increased the expression levels of positive cell cycle regulators such as cyclin A and cyclin-dependent kinases (CDK) 2. In contrast, levels of CDK inhibitors p27KIP1 and p57KIP2 were reduced upon treatment with AQ. Similar to the in vitro results, RT-qPCR analysis of AQ-administered mice brains revealed an increase in the levels of markers of cell cycle progression, PCNA, MCM5, and Cdc25a. Finally, AQ administration resulted in decreased p27KIP1 and increased CDK2 levels in the dentate gyrus of the mouse hippocampus, as quantified immunohistochemically. Our results demonstrate that the pharmacological stimulation of Nurr1 in adult hNSCs by AQ promotes the cell cycle by modulating cell cycle-related molecules.

Therapeutic targeting of the E3 ubiquitin ligase SKP2 in T-ALL

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Abstract NT-111: THE ANTIPROGESTIN/ANTIGLUCOCORTICOID MIFEPRISTONE AND THE HIV PROTEASE INHIBITOR NELFINAVIR CAUSE ENDOPLASMIC RETICULUM STRESS AND POTENTIATE THE TOXICITY OF PROTEASOME INHIBITION IN HIGH-GRADE SEROUS EPITHELIAL OVARIAN CANCER CELLS

Abstract Epithelial ovarian cancer (EOC) is a fatal disease due to late diagnosis and lack of effective long-term treatment(s). Since the introduction of debulking surgery and platinum (Pt)-taxane (Tx) therapy over 30 yrs. ago, there has been no significant breakthrough impacting the overall survival of these patients. Though over 70% of diagnosed women respond to front-line standard of care with remission, the disease hides as microscopic (minimal residual) within the abdominal cavity for about 18-24 months (mo.), recurring thereafter with a phenotype usually not responsive to current chemotherapeutic agents. As patients are left without treatment between remission and recurrence, our research initiative is to develop a consolidation therapy for chronic use after standard of care. In this work we explored whether such consolidation therapy could be developed from two simultaneous strategies: proteasome inhibition and aggravation of the stress of the endoplasmic reticulum (ER). The rationale for this combination therapy is that malignant cells are more susceptible to the toxicity of proteasome inhibition and operate with increased expression of ER stress-related proteins—coined as unfolded protein response (UPR) addiction—allowing cancer cells to survive in a hostile environment of reduced nutrients, acidosis, energy deficiency, and low oxygen tension (hypoxia). We hypothesized that simultaneous ER stress aggravation and blockage of the proteolytic capacity of the proteasome should cause sufficient ER stress to tilt cells to a death fate. We report that antiprogestin/antiglucocorticoid mifepristone (MF) as well as HIV protease inhibitor nelfinavir (NFV) enhanced the stress of the ER in EOC cells of high-grade serous origin (HGSOC), which is the most aggressive subtype of EOC, represents the majority of cases of EOC, and causes 2/3 of all deaths from this disease. We demonstrated, in HGSOC cells, that both MF and NFV cause cell cycle arrest associated with increased expression of cyclin dependent kinase inhibitor p27kip1, while triggering the UPR in a dose-dependent manner assessed by increased subrogate ER stress biomarkers GRP78, ATF4, and CHOP. We also discovered that blocking the ubiquitin proteasome system (UPS) using cytostatic concentrations of the proteasome inhibitor bortezomib (BZ) causes accumulation of poly-ubiquitinated proteins and further activation of the UPR. More importantly, when we combined BZ with either MF or NFV, we observed a potentiation of the action of BZ leading to EOC lethality. These results suggest that targeting the ER stress-associated protein quality control machinery with either an antiprogestin/antiglucocorticoid agent or an HIV protease inhibitor may provide an alternative chronic treatment approach against HGSOC after standard of care. In addition, the data provide the rationale for rapid repurposing to treating HGSOC, of three drugs clinically approved for other uses, as their systemic toxicities have already been assessed. Citation Format: Mahbuba Subeha, Lei Zhang, Alicia Goyeneche, Carlos Telleria. THE ANTIPROGESTIN/ANTIGLUCOCORTICOID MIFEPRISTONE AND THE HIV PROTEASE INHIBITOR NELFINAVIR CAUSE ENDOPLASMIC RETICULUM STRESS AND POTENTIATE THE TOXICITY OF PROTEASOME INHIBITION IN HIGH-GRADE SEROUS EPITHELIAL OVARIAN CANCER CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-111.

Invadopodia-mediated ECM degradation is enhanced in the G1 phase of the cell cycle.

The process of tumor cell invasion and metastasis includes assembly of invadopodia, protrusions capable of degrading the extracellular matrix (ECM). The effect of cell cycle progression on invadopodia has not been elucidated. In this study, by using invadopodia and cell cycle fluorescent markers, we show in 2D and 3D cultures, as well as in vivo, that breast carcinoma cells assemble invadopodia and invade into the surrounding ECM preferentially during the G1 phase. The expression (MT1-MMP, also known as MMP14, and cortactin) and localization (Tks5; also known as SH3PXD2A) of invadopodia components are elevated in G1 phase, and cells synchronized in G1 phase exhibit significantly higher ECM degradation compared to the cells synchronized in S phase. The cyclin-dependent kinase inhibitor (CKI) p27kip1 (also known as CDKN1B) localizes to the sites of invadopodia assembly. Overexpression and stable knockdown of p27kip1 lead to contrasting effects on invadopodia turnover and ECM degradation. Taken together, these findings suggest that expression of invadopodia components, as well as invadopodia function, are linked to cell cycle progression, and that invadopodia are controlled by cell cycle regulators. Our results caution that this coordination between invasion and cell cycle must be considered when designing effective chemotherapies.

Sodium nitroprusside attenuates hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats through the inhibition of overexpression of AT1 receptor, cell cycle proteins, and c-Src/growth factor receptor signaling pathways.

We recently showed that sodium nitroprusside (SNP), a NO donor, attenuated hypertension in spontaneously hypertensive rats (SHR). Since hypertension is associated with enhanced proliferation and hypertrophy of vascular smooth muscle cells (VSMC), the present study examines whether in vivo treatment of SHR with SNP could also inhibit the augmented proliferation of VSMC and explore the signaling mechanisms. Treatment of 8 week old SHR and Wistar Kyoto rats with SNP twice a week for 2 weeks inhibited the enhanced proliferation of VSMC from SHR, the enhanced expression of angiotensin II type 1 (AT1) receptor, and enhanced activation of c-Src and growth factor receptors and ERK1/2 signaling pathways. In addition, SNP also inhibited the overexpression of cell cycle proteins including cyclins D1, Cdk4, and phosphorylated pRB and restored the downregulated Cdk inhibitors p21Cip1 and p27Kip1 expression towards control levels. Furthermore, SNP-induced inhibition of enhanced levels of the AT1 receptor and enhanced proliferation was reversed by L-NAME, an inhibitor of nitric oxide synthase. These results suggest that the SNP-induced antiproliferative effect may be mediated through the inhibition of enhanced expression of the AT1 receptor, cell cycle proteins and activation of c-Src, growth factor receptors, and MAP kinase signaling.

Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells

The tight junctional pore-forming protein claudin-2 (CLDN-2) mediates paracellular Na+ and water transport in leaky epithelia and alters cancer cell proliferation. Previously, we reported that tumor necrosis factor-α time-dependently alters CLDN-2 expression in tubular epithelial cells. Here, we found a similar expression pattern in a mouse kidney injury model (unilateral ureteral obstruction), consisting of an initial increase followed by a drop in CLDN-2 protein expression. CLDN-2 silencing in LLC-PK1 tubular cells induced activation and phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1), leading to Ras homolog family member A (RHOA) activation. Silencing of other claudins had no such effects, and re-expression of an siRNA-resistant CLDN-2 prevented RHOA activation, indicating specific effects of CLDN-2 on RHOA. Moreover, kidneys from CLDN-2 knockout mice had elevated levels of active RHOA. Of note, CLDN-2 silencing reduced LLC-PK1 cell proliferation and elevated expression of cyclin-dependent kinase inhibitor P27 (P27KIP1) in a GEF-H1/RHOA-dependent manner. P27KIP1 silencing abrogated the effects of CLDN-2 depletion on proliferation. CLDN-2 loss also activated myocardin-related transcription factor (MRTF), a fibrogenic RHOA effector, and elevated expression of connective tissue growth factor and smooth muscle actin. Finally, CLDN-2 down-regulation contributed to RHOA activation and smooth muscle actin expression induced by prolonged tumor necrosis factor-α treatment, because they were mitigated by re-expression of CLDN-2. Our results indicate that CLDN-2 suppresses GEF-H1/RHOA. CLDN-2 down-regulation, for example, by inflammation, can reduce proliferation and promote MRTF activation through RHOA. These findings suggest that the initial CLDN-2 elevation might aid epithelial regeneration, and CLDN-2 loss could contribute to fibrotic reprogramming.

266Loss of pumilio 1 phosphorylation turns off the regulation of miRNA-221 on gene p27kip1 in the heart

Abstract Background MicroRNAs regulate gene expression post-transcriptionally via complementary sequence pairing of mRNA 3'UTR and miRNA seed region. MiR-221 downregulates p27kip1 Cyclin-dependent kinase inhibitor 1B (p27), a cell cycle inhibitor, through direct targeting. The down-regulation of p27 caused by miR-221 upregulation induces cancer cell proliferation and has been reported to be closely associated with human cancer metastasis. This regulation is dependent on the existence of pumilio 1 (PUM1). Meanwhile miR-221 mimics protect cardiac myocytes against ischemic injury. It is a potential therapeutic target for cardioprotection. We investigated whether miR-221 targeted p27 in the heart. Purpose To test the hypothesis that the regulation of miR-221 on p27 is abolished in the heart due to lack of RNA binding protein (RBP) PMU1 phosphorylation but not due to p27 3'UTR shortening. Methods Primary cardiac fibroblasts (cFB) were isolated and cultured under normoxia and hypoxia. After transfections of miR-221 mimics and mimic control (MC), cell proliferation was assessed by cell number, CCk-8 and Ki67 staining. In vivo rats undergoing left coronary artery ligation (MI) and Sham were treated with intravenous miR-221 mimics. Cells or cardiac tissue were collected to measure p27 and PUM1 by RT-qPCR and Western blot (WB). Full length p27 (coding plus 3'UTR sequence) and 3'UTR p27 sequence were quantified using specific primers. The ratio of 3'UTR/Full length was compared to indicate changes between groups. The ratios of 3'UTR/Full length were compared to indicate changes between groups. Results MiR-221 mimics significantly downregulated the expression of p27 mRNA and protein in cFB in both normoxia and hypoxia. P27 downregulation led to cFB proliferation, which could be abrogated by p27 overexpression. miR-221 mimic treatment increased cardiac miR-221 by ∼2–5 fold in Sham and ∼15 fold in MI. Whilst p27 expression was not affected in either Sham or MI hearts. Simultaneously α-SMA+ cells, an indication of cFB number and activation, were reduced by miR-221 mimic in Sham and MI heart. The ratios of p27 3'UTR/full length did not vary significantly between cultured cFB and heart (Sham and MI), indicating the 3'UTR length did not vary between cFB and the whole heart. PUM1 mRNA expression was stable in cFB and the heart. However, phosphorylated PUM1 was highly expressed in cFB but completely diminished in Sham and MI hearts. Conclusion MiR-221 downregulated p27 in primary cFB and induced cell proliferation in vitro. miR-221 could not regulate cardiac p27 expression nor increase cFB proliferation in vivo. MiR-221 induced p27 regulation was correlated with the presence or absence of phosphorylated PUM1. For the first time, we demonstrated that loss of phosphorylated PUM1 turns off the regulation of miRNA-221 on gene p27 in the heart. The mechanism of PUM1 phosphorylation warrant future investigation.

Toll Like Receptor 2 Genotypes and Intra-Coronary Stent Restenosis

AbstractBackground: Intra-coronary Stent Restenosis (ISR) is a common complication after Percutaneous Coronary Interven-tion (PCI). There is an association between many genes such as Toll Like Receptor 2 (TLR2), Adrenergic B2 Receptor (ADRB2), platelet glycoprotein IIIa, Nitric Oxide Synthase3 (NOS3), P2Y12 receptor (P2Y12), cyclin-dependent kinase inhibitor (p27kip1) and the process of intra-coronary stent restenosis. Study of the genetic polymorphisms of these genes is very important to detect which is protective against intra-coronary stent restenosis, and which is risky for development of this problem. Our study selected TLR2 gene to detect its different genotypes involved in the problem of ISR.Aim of Study: To find the association between TLR2 genotypes, polymorphisms and Intra-coronary Stent Restenosis (ISR) complication after PCI procedures.Patients and Methods: The study included 200 patients with previous coronary revascularization by stent implantation. All patients were re-admitted to coronary angiography because of objective evidence of myocardial ischemia. The patients were classified into two groups: Group (A) included 100 patients who developed ISR and Group (B) included 100 patients who did not develop ISR. The two groups were compared regarding distribution of TLR2 genotypes and alleles.Results: 78% of patients with (GG) genotype, 14% of patients with (GA) genotype and 7% of patients with (AA) genotype developed ISR. There was no association between TLR2 (GA), (AA) genotypes and risk of ISR when compared by (GG) wild homozygous genotype (p>0.05, OR=1.2). We also did not find any association between TLR2 gene alleles and risk of ISR complication (p>0.05, OR=0.9).Conclusion: There was no association between different TLR2 genotypes, alleles and risk of ISR development.

RUNX3 Promotes the Tumorigenic Phenotype in KGN, a Human Granulosa Cell Tumor-Derived Cell Line.

Granulosa cell tumors of the ovary (GCT) are the predominant type of ovarian sex cord/stromal tumor. Although prognosis is generally favorable, the outcome for advanced and recurrent GCT is poor. A better understanding of the molecular pathogenesis of GCT is critical to developing effective therapeutic strategies. Here we have examined the potential role of the runt-related transcription factor RUNX3. There are only two GCT cell lines available. While RUNX3 is silenced in the GCT cell line KGN cells, it is highly expressed in another GCT cell line, COV434 cells. Re-expression of RUNX3 promotes proliferation, anchorage-independent growth, and motility in KGN cells in vitro and tumor formation in mice in vivo. Furthermore, expression of a dominant negative form of RUNX3 decreases proliferation of COV434 cells. To address a potential mechanism of action, we examined expression of cyclin D2 and the CDK inhibitor p27Kip1, two cell cycle regulators known to be critical determinants of GCT cell proliferation. We found that RUNX3 upregulates the expression of cyclin D2 at the mRNA and protein level, and decreases the level of the p27Kip1 protein, but not p27Kip1 mRNA. In conclusion, we demonstrate that RUNX proteins are expressed in GCT cell lines and human GCT specimens, albeit at variable levels, and RUNX3 may play an oncogenic role in a subset of GCTs.