Induction Of Cyclin D1 Expression Research Articles

Abstract 1778: NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer

Abstract Genomic loss of in Neurofibromin 1 (NF1) is a recurrent mechanism driving metastatic progression and resistance to endocrine therapy in ER-positive breast cancer, via activation of the MAPK pathway, induction of cyclin D1 expression and modulation of estrogen receptor signaling. To identify specific mediators of tumor growth in NF1 mutant cancers, we performed a genetic knockout (KO) screen in isogenic WT and NF1 KO breast cancer cells. After employing stringent cutoff criteria, we found 28 genes that are specifically essential for NF1 KO cells but not WT cells. Among these genes, NR2F2, encoding an orphan nuclear receptor, was one of the top candidates. We validated that its loss significantly impaired the growth of multiple models of NF1 null, ER+ breast cancer. Loss of NF1 leads to the hyperactivation of Ras which sequentially activates multiple effector pathways, including the MAPK pathway and the PI3K-AKT pathway. We found that MAPK activation upon NF1 loss led to elevated NR2F2 expression levels. A negative correlation between NF1 and NR2F2 expression was further confirmed in public gene expression databases. These data suggested that induction of NR2F2 expression might be one mechanism that mediates endocrine resistance caused by NF1 loss. Consistently, we found that exogenous expression of NR2F2 could confer endocrine resistance while its knockout hypersensitized ER+ breast cancer cells to endocrine therapies and significantly attenuated NF1 loss induced resistance. RNA-seq analysis revealed that estrogen response gene sets were among the most enriched signatures upon NF1 KO, NR2F2 over-expression (OE) or NR2F2 KO. NF1 KO or NR2F2 OE decreased the expression of early estrogen response genes and impaired their E2-induced expression, most of which could be rescued by NR2F2 KO, suggesting a regulatory effect of NR2F2 on ER transcriptional activity. To further define these mechanisms, ongoing integrative analyses of NR2F2 qPlex-RIME, ER and NR2F2 ChIP-Sequencing and ATAC-Sequencing will be presented. Taken together, our studies identify NR2F2 as a MAPK driven transcript whose induction mediates endocrine resistance, and may serve as a novel therapeutic target to overcome anti-estrogen resistant breast cancer growth. Citation Format: Yanyan Cai, Peihua Zhao, Fan Wu, Hong Shao, Pedram Razavi, Guotai Xu, Maurizio Scaltriti, Sarat Chandarlapaty. NR2F2 mediates cell growth and endocrine resistance in NF1 loss, ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1778.

Identification of cyclin D1 as a major modulator of 3-nitropropionic acid-induced striatal neurodegeneration

Mitochondria dysfunction occurs in the aging brain as well as in several neurodegenerative disorders and predisposes neuronal cells to enhanced sensitivity to neurotoxins. 3-nitropropionic acid (3-NP) is a naturally occurring plant and fungal neurotoxin that causes neurodegeneration predominantly in the striatum by irreversibly inhibiting the tricarboxylic acid respiratory chain enzyme, succinate dehydrogenase (SDH), the main constituent of the mitochondria respiratory chain complex II. Significantly, although 3-NP-induced inhibition of SDH occurs in all brain regions, neurodegeneration occurs primarily and almost exclusively in the striatum for reasons still not understood.In rodents, 3-NP-induced striatal neurodegeneration depends on the strain background suggesting that genetic differences among genotypes modulate toxicant variability and mechanisms that underlie 3-NP-induced neuronal cell death. Using the large BXD family of recombinant inbred (RI) strains we demonstrate that variants in Ccnd1 - the gene encoding cyclin D1 - of the DBA/2 J parent underlie the resistance to 3-NP-induced striatal neurodegeneration. In contrast, the Ccnd1 variant inherited from the widely used C57BL/6 J parental strain confers sensitivity. Given that cellular stress triggers induction of cyclin D1 expression followed by cell-cycle re-entry and consequent neuronal cell death, we sought to determine if the C57BL/6 J and DBA/2 J Ccnd1 variants are differentially modulated in response to 3-NP. We confirm that 3-NP induces cyclin D1 expression in striatal neuronal cells of C57BL/6 J, but this response is blunted in the DBA/2 J. We further show that striatal-specific alternative processing of a highly conserved 3′UTR negative regulatory region of Ccnd1 co-segregates with the C57BL/6 J parental Ccnd1 allele in BXD strains and that its differential processing accounts for sensitivity or resistance to 3-NP. Our results indicate that naturally occurring Ccnd1 variants may play a role in the variability observed in neurodegenerative disorders involving mitochondria complex II dysfunction and point to cyclin D1 as a possible therapeutic target.

Cyclin K interacts with β-catenin to induce Cyclin D1 expression and facilitates tumorigenesis and radioresistance in lung cancer.

Rationale: Radioresistance remains the major cause of local relapse and distant metastasis in lung cancer. However, the underlying molecular mechanisms remain poorly defined. This study aimed to investigate the role and regulatory mechanism of Cyclin K in lung cancer radioresistance.Methods: Expression levels of Cyclin K were measured by immunohistochemistry in human lung cancer tissues and adjacent normal lung tissues. Cell growth and proliferation, neutral comet and foci formation assays, G2/M checkpoint and a xenograft mouse model were used for functional analyses. Gene expression was examined by RNA sequencing and quantitative real-time PCR. Protein-protein interaction was assessed by immunoprecipitation and GST pull-down assays.Results: We report that Cyclin K is frequently overexpressed and correlates with poor prognosis in lung cancer patients. Functionally, we demonstrate that Cyclin K depletion results in reduced proliferation, defective G2/M checkpoint and enhanced radiosensitivity in lung cancer. Mechanistically, we reveal that Cyclin K interacts with and promotes the stabilization of β-catenin protein, thereby upregulating the expression of Cyclin D1. More importantly, we show that Cyclin D1 is the major effector that mediates the biological functions of Cyclin K in lung cancer.Conclusions: These findings suggest that Cyclin K positively modulates the β-catenin/Cyclin D1 axis to promote tumorigenesis and radioresistance in lung cancer, indicating that Cyclin K may represent a novel attractive biomarker for lung cancer radiotherapy.

Leukotriene B4 induces proliferation of rat pulmonary arterial smooth muscle cells via modulating GSK-3β/β-catenin pathway

Leukotriene B4 (LTB4) has been found to contribute to pulmonary arterial smooth muscle cells (PASMCs) proliferation and pulmonary arterial remodeling therefore the development of pulmonary arterial hypertension (PAH). Yet, the underlying molecular mechanisms remain poorly understood. The present study aims to address this issue. Our results demonstrate that LTB4 dose- and time-dependently induced proliferation of primary cultured rat PASMCs, this was accompanied with the activation of phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, and consequent inactivation of glycogen synthase kinase-3β (GSK-3β), up-regulation of β-catenin and induction of cyclin D1 expression. The presence of PI3K inhibitor (LY294002) or MEK inhibitor (U0126) or prior silencing of β-catenin with siRNA suppressed LTB4-induced cyclin D1 up-regulation and PASMCs proliferation. In addition, inactivation or lack of GSK-3β up-regulated β-catenin and cyclin D1 in PASMCs. Taken together, our study indicates that activation of PI3K/Akt and ERK1/2 pathways mediates LTB4-induced PASMCs proliferation by modulating GSK-3β/β-catenin/cyclin D1 axis and suggests that targeting this pathway might have potential value in alleviating vascular remodeling and benefit PAH.

ProNGF Is a Cell-Type-Specific Mitogen for Adult Hippocampal and for Induced Neural Stem Cells.

The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR , as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223-1237.

Analysis of hard tissue regeneration and Wnt signalling in dental pulp tissues after direct pulp capping with different materials.

To investigate the involvement of Wnt signalling and cell cycle regulation in hard tissue formation after pulp capping with several materials in a rat molar pinpoint exposure model. Thirty-two rat molar pulps were mechanically exposed and assigned to 4 groups according to the pulp capping materials used Ca(OH)2 , mineral trioxide aggregate (MTA), Biodentine™ and an untreated control group. After 4weeks, the teeth were collected for microcomputed tomography to quantify reparative dentine formation. Histological analysis was then performed to evaluate the quality of the reparative dentine and the dental pulp tissue inflammatory reaction. Cyclin D1 and β-catenin expression was examined using immunofluorescence staining. The Kruskal-Wallis followed by Dunn's multiple comparison test was performed to determine significant differences. The exposed dental pulps treated with Ca(OH)2 , MTA and Biodentine™ exhibited reparative dentine formation near the exposure site. Fibrous tissues adjacent to the exposure site were observed in the untreated group. The microcomputed tomography evaluation of MTA and Biodentine™ groups revealed significantly greater reparative dentine formation compared with the control group (P=0.0032 in the MTA group and P=0.05 in the BiodentineTM group). From histological evaluations, the BiodentineTM group exhibited significantly greater reparative dentine formation grading compared with the control group (P=0.0152). The pulp tissues treated with Ca(OH)2 and Biodentine™ exhibited a lower inflammatory score compared with those of the untreated control (P=0.0291 in the Ca(OH)2 and P=0.0110 in the BiodentineTM group). Ca(OH)2 , MTA and Biodentine™ induced cyclin D1 expression in the dental pulp tissues adjacent to the reparative dentine. Moreover, the Biodentine™-treated defects demonstrated β-catenin expression in the pulp tissue adjacent to the newly formed reparative dentine, which was not observed with the other materials. All test materials promoted dentine bridge formation and stimulated cyclin D1 expression. The favourable outcome after direct pulp capping with Biodentine™ involved Wnt/β-catenin signalling. However, Wnt/β-catenin signalling did not participate in the mechanism by which Ca(OH)2 and MTA promoted reparative dentine formation.

Pigment epithelium-derived factor peptide promotes limbal stem cell proliferation through hedgehog pathway.

Expansion of limbal epithelial stem cells (LSCs) is crucial for the success of limbal transplantation. Previous studies showed that pigment epithelium‐derived peptide (PEDF) short peptide 44‐mer could effectively expand LSCs and maintain them in a stem‐cell state, but the mechanism remained unclear. In the current study, we found that pharmacological inhibition of Sonic Hedgehog (SHh) activity reduced the LSC holoclone number and suppressed LSC proliferation in response to 44‐mer. In mice subjected to focal limbal injury, 44‐mer facilitated the restoration of the LSC population in damaged limbus, and such effect was impeded by the SHh or ATGL (a PEDF receptor) inhibitor. Furthermore, we showed that 44‐mer increased nuclear translocation of Gli1 and Gli3 in LSCs. Knockdown of Gli1 or Gli3 suppressed the ability of 44‐mer to induce cyclin D1 expression and LSC proliferation. In addition, ATGL inhibitor suppressed the 44‐mer‐induced phosphorylation of STAT3 at Tyr705 in LSC. Both inhibitors for ATGL and STAT3 attenuated 44‐mer‐induced SHh activation and LSC proliferation. In conclusion, our data demonstrate that SHh‐Gli pathway driven by ATGL/STAT3 signalling accounts for the 44‐mer‐mediated LSC proliferation.

Up-regulated WDR5 promotes gastric cancer formation by induced cyclin D1 expression.

Gastric cancer (GC) is the fourth common cancer and second leading cause of cancer-related mortality in the world. WD repeat domain 5 (WDR5) has been identified that its functions as an important role in various biological functions through the epigenetic regulation of gene transcription. However, the oncogenic effect of WDR5 in gastric cancer remains largely unknown. In this study, we investigated the role of WDR5 in gastric cancer genesis. We found that WDR5 expression is increased in gastric cancer patients. Through survival analysis, we found that high expression of WDR5 is associated with high risk gastric cancer; patients who with WDR5 high expression have poor survival rate compared with those who with WDR5 low expression. To make further investigation, we identified that WDR5 is targeted for cell cycle arrest by the Cyclin D1 in a process that is regulated by H3K4me3. Moreover, over-expression of WDR5 promotes cell proliferation, induces S/G2/M arrest in cell cycle, and promotes the expression of WDR5 targets, as well as that of H3K4me3 on the promoter of its targets. Inversely, WDR5 knockdown by shRNA inhibits cell proliferation, reverses S/G2/M arrest in cell cycle, and suppresses the expression of WDR5 targets, as well as that of H3K4me3. We also observed the positive correlation of WDR5 expression with its target in the cohort study of gastric patients. Taken together, our data reveal that WDR5 may have oncogenic effect and WDR5-mediated H3K4 methylation plays an important role in gastric cancer.

Transcription activated p73-modulated cyclin D1 expression leads to doxorubicin resistance in gastric cancer.

Gastric cancer (GC) is one of the leading types of cancer in terms of mortality cases worldwide. Doxorubicin (Dox), a common chemotherapy drug, is frequently used to treat GC; however, acquired resistance to Dox hinders the chemotherapeutic outcome and causes shorter survival in GC patients. Several Dox-resistant GC cell lines, including SGC7901, SNU-1 and SNU-5 were generated to investigate the mechanism of Dox resistance in GC. Various methods were used to test the response of Dox-resistant GC cells and parental cells, including flow cytometry, Cell Counting kit-8 assay, reverse transcription polymerase chain reaction and western blot analysis. In the present study, various Dox-resistant cells presented reduced apoptosis and cell cycle arrest in response to Dox treatment. Western blot results revealed that cyclin D1 was upregulated in Dox-resistant cells, whereas inhibition or depletion of cyclin D1 re-sensitized the resistant cells to Dox treatment, which indicated that the induction of cyclin D1 expression was a result of the Dox resistance in GC cells. Furthermore, it was observed that a transcription activated form of p73 (TAp73), is the upstream modulator of cyclin D1, manipulating the cyclin D1 transcription with the assistance of activator protein 1 (AP-1). Overall, the present study data provided a rational strategy to overcome the Dox resistance in GC treatment by inhibiting cyclin D1 expression.

MicroRNA-328 inhibits cervical cancer cell proliferation and tumorigenesis by targeting TCF7L2

microRNAs (miRNAs) play a vital role in tumor development and progression. In this study, we aimed to determine the expression and biological roles of miR-328 in cervical cancer and identify its direct target gene. Our data showed that miR-328 was significantly downregulated in human cervical cancer tissues and cells. Re-expression of miR-328 inhibited cervical cancer cell proliferation and colony formation in vitro and suppressed the growth of xenograft tumors in vivo. Bioinformatic analysis predicted TCF7L2 (an essential effector of canonical Wnt signaling) as a target gene of miR-328, which was confirmed by luciferase reporter assays. Enforced expression of miR-328 led to a decline in the expression of endogenous TCF7L2 in cervical cancer cells. In cervical cancer tissues, TCF7L2 protein levels were negatively correlated with miR-328 expression levels (r = −0.462, P = 0.017). Small interfering RNA-mediated knockdown of TCF7L2 significantly impaired the proliferation and colony formation of cervical cancer cells. Ectopic expression of a miRNA-resistant form of TCF7L2 significantly reversed the growth suppressive effects of miR-328 on cervical cancer cells, which was accompanied by induction of cyclin D1 expression. Taken together, our results provide first evidence for the growth suppressive activity of miR-328 in cervical cancer, which is largely ascribed to downregulation of TCF7L2. Restoration of miR-328 may have therapeutic potential in cervical cancer.

Two inhibitory systems and CKIs regulate cell cycle exit of mammalian cardiomyocytes after birth

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which they exit their cell cycle after birth, and the exit is maintained. Previously, we showed that two inhibitory systems (the G1-phase inhibitory system: repression of cyclin D1 expression; the M-phase inhibitory system: inhibition of CDK1 activation) maintain the cell cycle exit of mouse adult cardiomyocytes. We also showed that two CDK inhibitors (CKIs), p21Cip1 and p27Kip1, regulate the cell cycle exit in a portion of postnatal cardiomyocytes. It remains unknown whether the two inhibitory systems are involved in the cell cycle exit of postnatal cardiomyocytes and whether p21Cip1 and p27Kip1 also inhibit entry to M-phase. Here, we showed that more than 40% of cardiomyocytes entered an additional cell cycle by induction of cyclin D1 expression at postnatal stages, but M-phase entry was inhibited in the majority of cardiomyocytes. Marked cell cycle progression and endoreplication were observed in cardiomyocytes of p21Cip1 knockout mice at 4 weeks of age. In addition, tri- and tetranucleated cardiomyocytes increased significantly in p21Cip1 knockout mice. These data showed that the G1-phase inhibitory system and two CKIs (p21Cip1 and p27Kip1) inhibit entry to an additional cell cycle in postnatal cardiomyocytes, and that the M-phase inhibitory system and p21Cip1 inhibit M-phase entry of cardiomyocytes which have entered the additional cell cycle.

Abstract 2707: Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3

Abstract The MDM2-p53 pathway plays a prominent role in various cancer pathogenesis. Nutlin-3, a small-molecule antagonist of MDM2, inhibits proliferation in cancer cells with wild-type p53 by blocking the interaction of MDM2 and p53. However, different cancer cells differ greatly sensitivity of Nutlin-3 because of MDM2 amplification and/or p53 mutation status. Herein, we evaluate the sensitivity of Nutlin-3 in different cancer cell lines including four mesothelioma cell lines, one breast cancer line, and one leiomyosarcoma cell line with nonmutant p53 expression, two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one control mesothelioma cell line JMN1B harboring p53 point mutation. Unexpectedly, Nutlin-3 treatment induced cyclin D1 expression accompanied with inhibition of MDM2-p53 interaction in nonmutant cancer cell lines. Additive effects were obtained through a coordinated attack on MDM2-p53 interaction and cyclin D1, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that lentiviral mediated cyclin D1 knockdown or drug inhibition, and block of MDM2-p53 interaction, in cancer cell lines containing wild-type p53, induced greater anti-proliferative effects, compared to either intervention alone. Further results demonstrated that upregulation of cyclin D1 after Nutlin-3 treatment to some extent was dependent on expression and ubiquitin E3-ligase activity of MDM2. These compelling anti-proliferative responses indicate that combination inhibition of cyclin D1 and MDM2-p53 interaction warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53. Citation Format: Wenbin Ou, Weicai Chen, Jiaqing Zhu, Grant Eilers, Xuhui Li, Peipei Yang, Hailong Li, Fanguo Meng, Jonathan Fletcher. Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3. [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 2707. doi:10.1158/1538-7445.AM2015-2707

Daxx and TCF4 interaction links to oral squamous cell carcinoma growth by promoting cell cycle progression via induction of cyclin D1 expression.

ObjectivesDeath domain-associated protein (Daxx) has been recently implicated as a positive factor in ovarian cancer and prostate cancer, but the role of Daxx in oral squamous cell carcinoma (OSCC) has never been addressed. Herein, we investigate the expression and function of Daxx in OSCC.Materials and methodsRT-quantitative PCR, Western blotting, and immunohistochemistry were used to evaluation of the expression of Daxx in human OSCC cell lines and clinical surgical specimens. Short hairpin RNA targeting Daxx was transduced by lentivirus infection to knockdown the expression of Daxx in SAS and SCC25 cell lines, and the influence of this knockdown was evaluated by analyzing the growth and the cell cycle in transduced cells. Immunoprecipitation and sequential chromatin immunoprecipitation-quantitative PCR were used to analyze the associations between Daxx, TCF4, and cyclin D1 promoter. Xenograft tumor model was used to evaluate the in vivo tumorigenicity of Daxx in OSCC.ResultsDaxx mRNA and protein expression are elevated in several OSCC cell lines and human OSCC samples in comparison to those in normal tissue. We further find that depletion of Daxx decreases OSCC cell growth activity through G1 cell cycle arrest. Daxx silencing reduces cyclin D1 expression via a Daxx-TCF4 interaction, whereas the Daxx depletion-mediated G1 arrest can be relieved by ectopic expression of cyclin D1. Moreover, we show that in OSCC clinical samples, the expression of Daxx is significantly correlated with that of cyclin D1.ConclusionOur data demonstrate the importance of Daxx in regulation of cyclin D1 expression and provide the first evidence that Daxx exhibits tumor-promoting activity in OSCC.Clinical relevanceDaxx plays an important role in malignant transformation of OSCC and may serves as a target for cancer prevention and treatment.

Sa1978 Overexpression of HER2 Converted the KRAS-Driven PanIN Lesions to IPMC Phenotype

Background: Enzymes that initiate tryptophan metabolism along the kynurenine pathway (KP) are commonly overexpressed in colorectal cancer (CRC). One of these enzymes, IDO1, has recently been shown to promote tumorigenesis in a mouse model of colitis associated cancer and promote nuclear translocation of β-catenin and proliferation in CRC cells. The current investigation is aimed at identifying the signaling pathways activated by various KP metabolites and determining if a similar effect exists in non-neoplastic primary colonic epithelial cells (CEC). Methods: In vitro models of human CRC using various CRC cell lines (HT29, HCT116 and SW480) as well as human colon primary CECs cultured in growth factor (Wnt3a, Noggin, R-Spondin) enriched media were used. All experiments were performed in serum and growth factor free culture media with or without the addition of KP metabolites such as Kynurenine (Kyn), Quinolinic Acid (QA) and Picolinic Acid (PA). Expression of mRNA and protein as well as their phosphorylation status was quantified at multiple time points, and cell proliferation was measured by MTT assay. Results: Addition of Kyn, QA and PA (100 μM) rapidly induced (5 min) phosphorylation of AKT (Ser473) and LRP (Ser1490), a required co-receptor for activating Wnt/β-catenin signaling. The KP metabolites also rapidly inactivated GSK-3β as indicated by phosphorylation at Ser9 and induced expression of nuclear β-catenin (P-Ser552). These changes were followed by an induction of CyclinD1 expression at 12-36 hours along with an increase in CRC cell proliferation after Kyn and QA application. However, with PA, no functional changes in proliferation were observed. In contrast to CRC cells, KP metabolites treatment of primary human CECs led to comparatively moderate and delayed changes in AKT, GSK-3β and βcatenin phosphorylation. Additionally, no change in LRP phosphorylation or effect on primary CEC proliferation was observed. Conclusions: KP metabolites induce rapid and reversible changes in the activation state of cellular machinery linked to CRC cell proliferation and neoplastic growth. While similarities exist between initial signaling events of KP metabolites, important distinctions were observed in their ultimate effects on proliferation of CRC and primary colonic epithelial cells. Together these observations highlight the role of the KP metabolites in β-catenin signaling and form the basis of future interrogation of this pathway for therapeutic targeting of colorectal cancer that may spare the normal epithelium.

Sa1977 Kynurenine Metabolic Pathway As a Rational Target for Colorectal Cancer: Defining Its Role in β-Catenin Signaling

Background: Enzymes that initiate tryptophan metabolism along the kynurenine pathway (KP) are commonly overexpressed in colorectal cancer (CRC). One of these enzymes, IDO1, has recently been shown to promote tumorigenesis in a mouse model of colitis associated cancer and promote nuclear translocation of β-catenin and proliferation in CRC cells. The current investigation is aimed at identifying the signaling pathways activated by various KP metabolites and determining if a similar effect exists in non-neoplastic primary colonic epithelial cells (CEC). Methods: In vitro models of human CRC using various CRC cell lines (HT29, HCT116 and SW480) as well as human colon primary CECs cultured in growth factor (Wnt3a, Noggin, R-Spondin) enriched media were used. All experiments were performed in serum and growth factor free culture media with or without the addition of KP metabolites such as Kynurenine (Kyn), Quinolinic Acid (QA) and Picolinic Acid (PA). Expression of mRNA and protein as well as their phosphorylation status was quantified at multiple time points, and cell proliferation was measured by MTT assay. Results: Addition of Kyn, QA and PA (100 μM) rapidly induced (5 min) phosphorylation of AKT (Ser473) and LRP (Ser1490), a required co-receptor for activating Wnt/β-catenin signaling. The KP metabolites also rapidly inactivated GSK-3β as indicated by phosphorylation at Ser9 and induced expression of nuclear β-catenin (P-Ser552). These changes were followed by an induction of CyclinD1 expression at 12-36 hours along with an increase in CRC cell proliferation after Kyn and QA application. However, with PA, no functional changes in proliferation were observed. In contrast to CRC cells, KP metabolites treatment of primary human CECs led to comparatively moderate and delayed changes in AKT, GSK-3β and βcatenin phosphorylation. Additionally, no change in LRP phosphorylation or effect on primary CEC proliferation was observed. Conclusions: KP metabolites induce rapid and reversible changes in the activation state of cellular machinery linked to CRC cell proliferation and neoplastic growth. While similarities exist between initial signaling events of KP metabolites, important distinctions were observed in their ultimate effects on proliferation of CRC and primary colonic epithelial cells. Together these observations highlight the role of the KP metabolites in β-catenin signaling and form the basis of future interrogation of this pathway for therapeutic targeting of colorectal cancer that may spare the normal epithelium.

Cyclin D1 represses gluconeogenesis via inhibition of the transcriptional coactivator PGC1α.

Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1–mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator–activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

The LIM-only protein FHL2 reduces vascular lesion formation involving inhibition of proliferation and migration of smooth muscle cells.

The LIM-only protein FHL2, also known as DRAL or SLIM3, has a function in fine-tuning multiple physiological processes. FHL2 is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells and conflicting data have been reported on the regulatory function of FHL2 in SMC phenotype transition. At present the function of FHL2 in SMCs in vascular injury is unknown. Therefore, we studied the role of FHL2 in SMC-rich lesion formation. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice showed accelerated lesion formation with enhanced Ki67 expression compared with wild-type (WT)-mice. Consistent with these findings, cultured SMCs from FHL2-KO mice showed increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. Overexpression of FHL2 in SMCs inhibited CyclinD1 expression and CyclinD1-knockdown blocked the enhanced proliferation of FHL2-KO SMCs. We also observed increased CyclinD1 promoter activity in FHL2-KO SMCs, which was reduced upon ERK1/2 inhibition. Furthermore, FHL2-KO SMCs showed enhanced migration compared with WT SMCs. In conclusion, FHL2 deficiency in mice results in exacerbated SMC-rich lesion formation involving increased proliferation and migration of SMCs via enhanced activation of the ERK1/2-CyclinD1 signaling pathway.

NFATc1 mediates Pak1 activation in the modulation of chemokine‐induced human aortic smooth muscle cell F‐actin stress fiber formation, migration, and proliferation and injury‐induced vascular wall remodeling (1158.5)

In addition to their role in cell proliferation, cyclin‐dependent kinases (CDKs) mediate cell migration; however the underlying mechanisms are not clear. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a time‐dependent but delayed manner. MCP1 also stimulated F‐actin stress fiber formation as well as the migration and proliferation of these cells and inhibition of Pak1 reversed these effects. MCP1 also induced cyclin D1 expression and CDK6/4 activities, and these effects were dependent on NFATc1 activation. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1‐induced Pak1 phosphorylation/activation resulting in decreased HASMC F‐actin stress fiber formation, migration and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. Depletion/inhibition of Rac1 levels/activation abrogated MCP1‐induced NFATc1‐cyclin D1‐CDK6‐CDK4‐Pak1 signaling, and thereby, decreased HASMC F‐actin stress fiber formation, migration, and proliferation. In addition, smooth muscle‐specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1‐dependent NFATc1‐mediated cyclin D1 expression and CDK6 activity mediate this effect in the modulation of MCP1‐induced HASMC migration and proliferation in vitro and vascular wall remodeling in vivo.Grant Funding Source: NHLBI

Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

Recent literature suggests that cyclin-dependent kinases (CDKs) mediate cell migration. However, the mechanisms were not known. Therefore, the objective of this study is to test whether cyclin/CDKs activate Pak1, an effector of Rac1, whose involvement in the modulation of cell migration and proliferation is well established. Monocyte chemotactic protein 1 (MCP1) induced Pak1 phosphorylation/activation in human aortic smooth muscle cells (HASMCs) in a delayed time-dependent manner. MCP1 also stimulated F-actin stress fiber formation in a delayed manner in HASMCs, as well as the migration and proliferation of these cells. Inhibition of Pak1 suppressed MCP1-induced HASMC F-actin stress fiber formation, migration, and proliferation. MCP1 induced cyclin D1 expression as well as CDK6 and CDK4 activities, and these effects were dependent on activation of NFATc1. Depletion of NFATc1, cyclin D1, CDK6, or CDK4 levels attenuated MCP1-induced Pak1 phosphorylation/activation and resulted in decreased HASMC F-actin stress fiber formation, migration, and proliferation. CDK4, which appeared to be activated downstream of CDK6, formed a complex with Pak1 in response to MCP1. MCP1 also activated Rac1 in a time-dependent manner, and depletion/inhibition of its levels/activation abrogated MCP1-induced NFATc1-cyclin D1-CDK6-CDK4-Pak1 signaling and, thereby, decreased HASMC F-actin stress fiber formation, migration, and proliferation. In addition, smooth muscle-specific deletion of NFATc1 led to decreased cyclin D1 expression and CDK6, CDK4, and Pak1 activities, resulting in reduced neointima formation in response to injury. Thus, these observations reveal that Pak1 is a downstream effector of CDK4 and Rac1-dependent, NFATc1-mediated cyclin D1 expression and CDK6 activity mediate this effect. In addition, smooth muscle-specific deletion of NFATc1 prevented the capacity of vascular smooth muscle cells for MCP-1-induced activation of the cyclin D1-CDK6-CDK4-Pak1 signaling axis, affecting their migration and proliferation in vitro and injury-induced neointima formation in vivo.

Abstract 307: Deficiency of Lim-only Protein Fhl2 Accelerates Neointima Formation After Carotid Artery Ligation

Rationale The LIM-only protein FHL2, also known as DRAL or SLIM3, has a regulatory function in many physiological processes and is expressed in the vessel wall in smooth muscle cells (SMCs) and endothelial cells, but not in macrophages. FHL2 regulates SMC phenotype, but its function in vascular injury is unknown. Objective To assess the role of FHL2 in SMC-rich lesion formation after vascular injury and to elucidate the underlying mechanism. Methods & Results Cultured aortic SMCs from FHL2-KO mice showed increased migration and increased proliferation through enhanced phosphorylation of extracellular-regulated kinase-1/2 (ERK1/2) and induction of CyclinD1 expression. In agreement with this, overexpression of FHL2 in SMCs reduced CyclinD1 expression. In addition, FHL2-KO SMCs showed enhanced expression of pro-inflammatory cytokines in a NFkB dependent manner. Consistent with these findings, NFkB activity is higher in FHL2-KO SMCs. In response to carotid artery ligation FHL2-deficient (FHL2-KO) mice developed accelerated lesion formation compared with wild-type (WT)-mice. Furthermore, FHL2-KO mice displayed high number of macrophages in lesions and enhanced expression of RANTES and stromal derived factor-1α (SDF-1α) in ligated carotid arteries. SDF-1α expression was also increased in plasma of FHL2-KO mice. Finally, FHL2-KO mice showed enhanced Ki67 expression in lesions compared with wild-type (WT)-mice. Conclusion FHL2 deficiency in mice results in an exacerbated neointima formation by enhanced proliferation and migration of SMCs, possibly regulated via ERK1/2 and NFkB pathway.