Cyclin D1 Accumulation Research Articles

A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

Complement factor B regulates cellular senescence and is associated with poor prognosis in pancreatic cancer

BackgroundThe interplay between cancer cells and stromal components, including soluble mediators released from cancer cells, contributes to the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we set out to identify key secreted proteins involved in PDAC progression.MethodsWe performed secretome analyses of culture media of mouse pancreatic intraepithelial neoplasia (PanIN) and PDAC cells using Stable Isotope Labeling by Amino acid in Cell culture (SILAC) with click chemistry and liquid chromatography-mass spectrometry (LC-MS/MS). The results obtained were verified in primary PDAC tissue samples and cell line models.ResultsComplement factor B (CFB) was identified as one of the robustly upregulated proteins, and found to exhibit elevated expression in PDAC cells compared to PanIN cells. Endogenous CFB knockdown by a specific siRNA dramatically decreased the proliferation of PDAC cells, PANC-1 and MIA PaCa-II. CFB knockdown induced increases in the number of senescence-associated-β-galactosidase (SA-β-gal) positive cells exhibiting p21 expression upregulation, which promotes cellular senescence with cyclinD1 accumulation. Furthermore, CFB knockdown facilitated downregulation of proliferating cell nuclear antigen and led to cell cycle arrest in the G1 phase in PDAC cells. Using immunohistochemistry, we found that high stromal CFB expression was associated with unfavorable clinical outcomes with hematogenous dissemination after surgery in human PDAC patients. Despite the presence of enriched CD8+ tumor infiltrating lymphocytes in the PDAC tumor microenvironments, patients with a high stromal CFB expression exhibited a significantly poorer prognosis compared to those with a low stromal CFB expression. Immunofluorescence staining revealed a correlation between stromal CFB expression in the tumor microenvironment and an enrichment of immunosuppressive regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We also found that high stromal CFB expression showed a positive correlation with high CD8+/Foxp3+ Tregs populations in PDAC tissues.ConclusionsOur data indicate that CFB, a key secreted protein, promotes proliferation by preventing cellular senescence and is associated with immunological tumor promotion in PDAC. These findings suggest that CFB may be a potential target for the treatment of PDAC.

Expression of PRIP, a phosphatidylinositol 4,5-bisphosphate binding protein, attenuates PI3K/AKT signaling and suppresses tumor growth in a xenograft mouse model

Cancer is characterized by uncontrolled proliferation resulting from aberrant cell cycle progression. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling, a regulatory pathway for the cell cycle, stabilizes cyclin D1 in the G1 phase by inhibiting the activity of glycogen synthase kinase 3β (GSK3β) via phosphorylation. We previously reported that phospholipase C-related catalytically inactive protein (PRIP), a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding protein, regulates PI3K/AKT signaling by competitively inhibiting substrate recognition by PI3K. Therefore, in this study, we investigated whether PRIP is involved in cell cycle progression.PRIP silencing in MCF-7 cells, a human breast cancer cell line, demonstrated PI(3,4,5)P3 signals accumulated at the cell periphery compared to that of the control. This suggests that PRIP reduction enhances PI(3,4,5)P3-mediated signaling. Consistently, PRIP silencing in MCF-7 cells exhibited increased phosphorylation of AKT and GSK3β which resulted in cyclin D1 accumulation. In contrast, the exogenous expression of PRIP in MCF-7 cells evidenced stronger downregulation of AKT and GSK3β phosphorylation, reduced accumulation of cyclin D1, and diminished cell proliferation in comparison to control cells. Flow cytometry analysis indicated that MCF-7 cells stably expressing PRIP attenuate cell cycle progression. Importantly, tumor growth of MCF-7 cells stably expressing PRIP was considerably prevented in an in vivo xenograft mouse model. In conclusion, PRIP expression downregulates PI3K/AKT/GSK3β-mediated cell cycle progression and suppresses tumor growth. Therefore, we propose that PRIP is a new therapeutic target for anticancer therapy.

3EZ, 20Ac-ingenol-induced Apoptosis in Chemoresistant Cancers With Cyclin D1 Accumulation.

The topoisomerase 1 catalytic inhibitor 3EZ, 20Ac-ingenol specifically induces apoptosis through the activation of ATR and the up-regulation of PTEN by enhancing the DNA damage response (DDR) in human B lymphoma (BALL-1) cells. The accumulation of cyclin D1 in cancer is known to be related to chemoresistance to DNA damage agents and nuclei of BALL-1 cells exhibit high levels of cyclin D1. However, 3EZ, 20Ac-ingenol effectively induced apoptosis of BALL-1 cells. Cell growth, protein levels, and apoptosis were determined by an MTT assay, immunoblotting and DNA fragmentation assay, respectively. 3EZ, 20Ac-ingenol strongly induced inhibition of cell proliferation and apoptosis in Jeko-1 and Panc-1 cell lines through the activation of tumor suppressor proteins and caspase 3. 3EZ, 20Ac-ingenol-induced apoptosis might occur in cells with cyclin D1 accumulation through enhancing DDR, regardless of the cancer cell type.

Wnt/β-catenin signaling pathway inhibits porcine reproductive and respiratory syndrome virus replication by enhancing the nuclear factor-κB-dependent innate immune response

The Wnt/β-catenin signaling pathway is an evolutionarily highly conserved signaling pathway related to the replication of various viruses. However, the interaction between the Wnt/β-catenin pathway and porcine reproductive and respiratory syndrome virus (PRRSV) is unknown. In the present study, we showed that PRRSV-infected Marc-145 and PAM cells expressed high levels of c-myc and cyclinD1 mRNA and accumulation of β-catenin in the nucleus. PRRSV nonstructural proteins (Nsps) 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by open reading frames (ORFs) 2b, 3, and 5 induced the activation of the Wnt pathway according to TOP/FOP luciferase reporter assay. But, Nsp5 inhibited the activation of the Wnt pathway. Pre-treatment with Wnt3a inhibited PRRSV replication in Marc-145 cells in a dose-dependent manner. Over-expression of β-catenin also inhibited PRRSV replication, while silencing of β-catenin by small hairpin RNA increased its replication in Marc-145 cells. Over-expression of β-catenin increased interferon regulatory factor (IRF)-3 expression and nuclear factor (NF)-κB phosphorylation, NF-κB and interferon-stimulated response element promoter activities, and interferon-β, DExD/H-box helicase 58 (DDX58), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-8 mRNA expression. Conversely, silencing β-catenin decreased phosphorylated IRF-3 and NF-κB, NF-κB and IFIT1 promoter activities, and IFN-β, DDX58, IFIT1, IL-1β, TNF-α, and IL-8 mRNA levels in Marc-145 cells. Co-immunoprecipitation and immunofluorescence colocalization analyses confirmed that β-catenin interacted with NF-κB in Marc-145 cells. In conclusion, PRRSV infection activates the Wnt/β-catenin signaling pathway via Nsps 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by ORFs 2b, 3, and 5. The Wnt/β-catenin pathway then inhibits PRRSV replication by enhancing the NF-κB-dependent innate immune response. These findings further our understanding of the role of the Wnt/β-catenin signaling pathway in regulating PRRSV replication and provide new insights into virus–host interactions.

HSulf‑1 and palbociclib exert synergistic antitumor effects on RB‑positive triple‑negative breast cancer.

Human sulfatase-1 (HSulf-1) is emerging as a novel prognostic biomarker in breast cancer. Previous studies demonstrated HSulf-1 to function as a negative regulator of cyclin D1 in breast cancer. Accumulating preclinical evidence is supporting the efficacy of cyclin-dependent kinase (CDK) 4/6 inhibitors against the luminal androgen receptor sub-type of triple-negative breast cancer (TNBC). It was therefore hypothesized that HSulf-1 may cooperate with CDK4/6 inhibitors to control cell cycle progression in breast cancer cells. HSulf-1 expression was found to be downregulated in TNBC tissues and cell lines compared with that in healthy tissues and non-breast cancer cell lines, respectively. High levels of HSulf-1 expression was also found to be associated with increased progression-free survival and overall survival in patients with TNBC. Functionally, it was demonstrated that HSulf-1 served as tumor suppressor in TNBC by inducing cell cycle arrest and apoptosis whilst inhibiting proliferation, epithelial-mesenchymal transition, migration and invasion. Subsequent overexpression of HSulf-1 coupled with treatment with the CDK4/6 inhibitor palbociclib exhibited a synergistic antitumor effect on retinoblastoma (RB)-positive TNBC. Further studies revealed the mechanism underlying this cooperative antiproliferative effect involved to be due to the prohibitive effects of HSulf-1 on the palbociclib-induced accumulation of cyclin D1 through AKT/STAT3 and ERK1/2/STAT3 signaling. Taken together, findings from the present study not only suggest that HSulf-1 may be a potential therapeutic target for TNBC, but also indicate that combinatorial treatment could be an alternative therapeutic option for RB-positive TNBC, which may open novel perspectives.

The PDE4 inhibitor CHF6001 affects keratinocyte proliferation via cellular redox pathways

Psoriasis is a skin disease characterized by abnormal keratinocyte proliferation and inflammation. Currently, there are no cures for this disease, so the goal of treatment is to decrease inflammation and slow down the associated rapid cell growth and shedding. Recent advances have led to the usage of phosphodiesterase 4 (PDE4) inhibitors for treatment of this condition. For example, apremilast is an oral, selective PDE4 inhibitor that is able to reduce skin inflammation and is Food and Drug Administration (FDA)-approved to treat adults with moderate to severe psoriasis and/or psoriatic arthritis. However, common target-related adverse events, including diarrhea, nausea, headache, and insomnia limit the usage of this drug. To circumvent these effects, the usage of PDE4 inhibitors specifically designed for topical treatment, such as CHF6001, may combine local anti-inflammatory activity with limited systemic exposure, improving tolerability. In this study, we showed that CHF6001, currently undergoing clinical development for COPD, suppresses human keratinocyte proliferation as assessed via BrdU incorporation. We also observed decreased re-epithelialization in a scratch-wound model after CHF6001 treatment. At the molecular level, CHF6001 inhibited translocation of phosphorylated NF-κB subunit p65, promoting loss of nuclear cyclin D1 accumulation and an increase of cell cycle inhibitor p21. Furthermore, CHF6001 decreased oxidative stress, measured by assessing lipid peroxidation (4-HNE adduct formation), through the inactivation of the NADPH oxidase.These results suggest that CHF6001 has the potential to treat skin disorders associated with hyperproliferative keratinocytes, such as psoriasis by targeting oxidative stress, abnormal re-epithelization, and inflammation.

Induction of endoplasmic reticulum stress and inhibition of colon carcinogenesis by the anti-helmintic drug rafoxanide.

Colorectal cancer (CRC) remains one of the leading causes of mortality worldwide. Drug repositioning is a promising approach for new cancer therapies, as it provides the opportunity to rapidly advance potentially promising agents into clinical trials. The FDA-approved anti-helminthic drug rafoxanide was recently reported to antagonize the oncogenic function of the BRAF V600E mutant protein, commonly found in CRCs, as well as to inhibit the proliferation of skin cancer cells. These observations prompted us to investigate the potential anti-cancer effects of rafoxanide in CRC models. We found rafoxanide inhibited proliferation in CRC cells, but not in normal colonic epithelial cells. Rafoxanide's anti-proliferative action was associated with marked reduction in cyclin D1 protein levels and accumulation of cells in the G0/G1 phase. These effects relied on selective induction of the endoplasmic reticulum stress (ERS) response in CRC cells and were followed by caspase-dependent cell death. Systemic administration of rafoxanide to Apcmin/+ mice induced to develop CRCs caused ERS activation, proliferation inhibition and apoptosis induction in the neoplastic cells. Collectively, our data suggest rafoxanide might be repurposed as an anti-cancer drug for the treatment of CRC.

Cellular Effects of Butyrate on Vascular Smooth Muscle Cells are Mediated through Disparate Actions on Dual Targets, Histone Deacetylase (HDAC) Activity and PI3K/Akt Signaling Network.

Vascular remodeling is a characteristic feature of cardiovascular diseases. Altered cellular processes of vascular smooth muscle cells (VSMCs) is a crucial component in vascular remodeling. Histone deacetylase inhibitor (HDACI), butyrate, arrests VSMC proliferation and promotes cell growth. The objective of the study is to determine the mechanism of butyrate-induced VSMC growth. Using proliferating VSMCs exposed to 5 mM butyrate, immunoblotting studies are performed to determine whether PI3K/Akt pathway that regulates different cellular effects is a target of butyrate-induced VSMC growth. Butyrate inhibits phosphorylation-dependent activation of PI3K, PDK1, and Akt, eliciting differential effects on downstream targets of Akt. Along with previously reported Ser9 phosphorylation-mediated GSK3 inactivation leading to stability, increased expression and accumulation of cyclin D1, and epigenetic histone modifications, inactivation of Akt by butyrate results in: transcriptional activation of FOXO1 and FOXO3 promoting G1 arrest through p21Cip1/Waf1 and p15INK4B upregulation; inactivation of mTOR inhibiting activation of its targets p70S6K and 4E-BP1 impeding protein synthesis; inhibition of caspase 3 cleavage and downregulation of PARP preventing apoptosis. Our findings imply butyrate abrogates Akt activation, causing differential effects on Akt targets promoting convergence of cross-talk between their complimentary actions leading to VSMC growth by arresting proliferation and inhibiting apoptosis through its effect on dual targets, HDAC activity and PI3K/Akt pathway network.

Estrogen Receptor Beta (ERβ) Mediated-CyclinD1 Degradation via Autophagy Plays an Anti-Proliferation Role in Colon Cells.

Dysfunction of autophagic degradation machinery causes tumorigenesis, including colorectal cancer (CRC). Overexpression of CyclinD1 in CRC has been reported. Recent evidence also suggests that ERβ deficiency is related to the pathogenesis of CRC. Very little is known, however, about the detailed molecular mechanisms underlying the relationship among ERβ, autophagy, and CyclinD1 in CRC. Here, results showed that ERβ played an anti-proliferation role in HCT116 through impairing cell cycle but not apoptosis. Additionally, CyclinD1 accumulation was increased in response to chloroquine (CQ) or in MEF Atg7 knockout cells. Further, ERβ could inhibit the mammalian target of rapamycin (mTOR) or activate Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) to promote autophagy in HCT116. In summary, these results indicate that ERβ-mediated CyclinD1 degradation can inhibit colon cancer cell growth via autophagy.

Loss of BRCA1 Spontaneously Induces the Tumorigenesis in Lacrimal Gland.

Environmental and genetic factors exert important influences on lifespan and neoplastic transformation. We have previously shown that spontaneous tumors form frequently in mice homozygous for a full-length Brca1 deletion. In general, mutations of BRCA1 are closely associated with induction of breast and ovarian cancers but are also known to contribute to the incidence of other cancers at a low frequency. Female Brca1-mutant mice (Brca1co/coMMTV-cre) were generated by crossing Brca1 conditional knockout mice and MMTV-cre mice, and the occurrence of lacrimal gland abnormalities and tumors was followed until mice reached 18 months of age. Lacrimal gland tumors, which occur at a very low frequency in the human population (1 per 1,000,000 per year), were detected in 7 cases of Brca1co/coMMTV-cre mice (2.75%) older than 9 months of age. None of seven mice exhibited any abnormality in the mammary gland including neoplasia, suggesting lacrimal gland tumor is spontaneously and independently formed. These tumors, which were detected in seven mutant mice that displayed exophthalmoses, were malignant, originated from epithelial cells, and were identified as acinic cell carcinoma by pathological analysis. Further analysis revealed that tumorigenesis was accompanied by the accumulation of cyclin D1 and decreased expression of the cellular oncogenes, c-Myc, c-Jun, and c-Raf. Tumors also exhibited rearrangement of cytoskeletal proteins, including β-catenin, keratin 5, and vimentin, depending on tumor progression. These results suggest that BRCA1 is involved in genetic stability of the lacrimal gland, providing new insight into genomic instability in organism maintenance and tumorigenesis of the lacrimal gland.

PI3K/Akt signaling transduction pathway, erythropoiesis and glycolysis in hypoxia (Review)

The purpose of this review is to summarize the research progress of PI3K/Akt signaling pathway in erythropoiesis and glycolysis. Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is activated by numerous genes and leads to protein kinase B (Akt) binding to the cell membrane, with the help of phosphoinositide-dependent kinase, in the PI3K/Akt signal transduction pathway. Threonine and serine phosphorylation contribute to Akt translocation from the cytoplasm to the nucleus and further mediates enzymatic biological effects, including those involved in cell proliferation, apoptosis inhibition, cell migration, vesicle transport and cell cancerous transformation. As a key downstream protein of the PI3K/Akt signaling pathway, hypoxia-inducible factor (HIF)-1 is closely associated with the concentration of oxygen in the environment. Maintaining stable levels of HIF-1 protein is critical under normoxic conditions; however, HIF-1 levels quickly increase under hypoxic conditions. HIF-1α is involved in the acute hypoxic response associated with erythropoietin, whereas HIF-2α is associated with the response to chronic hypoxia. Furthermore, PI3K/Akt can reduce the synthesis of glycogen and increase glycolysis. Inhibition of glycogen synthase kinase 3β activity by phosphorylation of its N-terminal serine increases accumulation of cyclin D1, which promotes the cell cycle and improves cell proliferation through the PI3K/Akt signaling pathway. The PI3K/Akt signaling pathway is closely associated with a variety of enzymatic biological effects and glucose metabolism.

A Pre-Clinical Large Animal Model of Sustained Liver Injury and Regeneration Stimulus

A feasible large animal model to evaluate regenerative medicine techniques is vital for developing clinical applications. One such appropriate model could be to use retrorsine (RS) together with partial hepatectomy (PH). Here, we have developed the first porcine model using RS and PH. RS or saline control was administered intraperitoneally to Göttingen miniature pigs twice, two weeks apart. Four weeks after the second dose, animals underwent PH. Initially, we tested different doses of RS and resection of different amounts of liver, and selected 50 mg/kg RS with 60% hepatectomy as our model for further testing. Treated animals were sacrificed 3, 10, 17 or 28 days after PH. Blood samples and resected liver were collected. Serum and liver RS content was determined by Liquid Chromatograph-tandem Mass Spectrometer. Blood analyses demonstrated liver dysfunction after PH. Liver regeneration was significantly inhibited 10 and 17 days after PH in RS-treated animals, to the extent of 20%. Histological examination indicated hepatic injury and regenerative responses after PH. Immunohistochemical staining demonstrated accumulation of Cyclin D1 and suppression of Ki-67 and PCNA in RS-treated animals. We report the development of the first large animal model of sustained liver injury with suppression of hepatic regeneration.

(\u2212)-Gochnatiolide B, synthesized from dehydrocostuslactone, exhibits potent anti-bladder cancer activity in vitro and in vivo

With limited success achieved in bladder cancer patient management, novel agents are in urgent need for the purpose of therapy and prevention. As a sesquiterpenoid dimmer isolated from Gochnatia pomculat, (−)-gochnatiolide B has been bio-mimetically synthesized in multiple steps with a poor yield, which heavily limited the further research and clinical application. Herein, (−)-gochnatiolide B was synthesized beginning with dehydrocostuslactone in four steps with a total yield of 26%. MTT assays showed that (−)-gochnatiolide B inhibited the growth of vast majority of human cancer cells especially bladder cancer cells. Mechanistically, (−)-gochnatiolide B induced the increased expression of pro-apoptotic proteins and the decreased expression of anti-apoptosis proteins and further resulted in apoptosis of T24 cells. (−)-Gochnatiolide B induced G1 arrest which associated with SKP2 downregulation, leading to p27/Kip1 accumulation and downregulation of cyclin D1 in T24 cells. Furthermore, in vivo studies showed that (−)-gochnatiolide B remarkably inhibited tumor growth by 81% compared with vehicle control. Taken together, (−)-gochnatiolide B exhibits inhibitory activity against bladder cancer cells both in vitro and in vivo by inducing apoptosis, which suggests that (−)-gochnatiolide B could be an important candidate compound for prevention and treatment of bladder cancer.

Abstract 617: Apolipoprotein E Receptor-2 Deficiency Accelerates Smooth Muscle Cell Senescence via Impairment of Protein Phosphatase-2-mediated Cytokinesis

Objective: Genome-wide association studies revealed apoE receptor-2 (apoER2) variants increase atherosclerosis risk in humans. ApoER2 deficiency in hypercholesterolemic Ldlr -/- mice also displayed accelerated atherosclerosis with more complex lesions and extensive lesion necrosis. While loss of apoER2 in macrophages contributes to the advanced lesion phenotype, apoER2 is also expressed in vascular smooth muscle cells (SMC). This study explores the role of apoER2 in SMC functions. Methods and Results: Freshly isolated aortic SMC from apoER2 knockout mice showed ~40% less proliferative capacity and displayed a significant reduction in their population doubling when compared to SMC from wild-type (WT) mice. Despite the reduced proliferative rate, apoER2-deficient SMC responded more robustly to TGF-β induced fibronectin synthesis, suggesting a senescence-associated secretory phenotype with increased fibrosis. Accelerated senescence of apoER2-deficient SMC was confirmed by cytochemical staining of β-galactosidase activity and by the increased number of multinucleated polyploidy cells compared to control. Western blot analysis of cell cycle proteins revealed the accumulation of cyclin B1, D1 and securin in apoER2-deficient SMC, indicating that loss of apoER2 does not affect cell cycle entry but promotes cell cycle arrest at the metaphase/anaphase. Increased levels of phosphorylated aurora kinases A/B/C were also observed in apoER2-deficient SMC, suggesting the impairment of protein phosphatases during cell cycle exit. Interestingly, co-immunoprecipitation experiments showed that apoER2 interacts with the catalytic subunit of protein phosphatase 2A (PP2A-C). In the absence of apoER2, PP2A-C failed to interact with cell-division cycle protein 20 (CDC20) thus resulting in inactive anaphase promoting complex (APC/C CDC20 ) and the impairment of cell cycle exit. Conclusions: This study identifies a novel function of the lipoprotein receptor apoER2 in SMC mitosis. The data indicate that apoER2 promotes the recruitment of PP2A-C to the anaphase complex, and the impairment of this process accelerates premature smooth muscle cell senescence similar to that observed in the aging vessel wall.

Protein C receptor stimulates multiple signaling pathways in breast cancer cells

The protein C receptor (PROCR) has emerged as a stem cell marker in several normal tissues and has also been implicated in tumor progression. However, the functional role of PROCR and the signaling mechanisms downstream of PROCR remain poorly understood. Here, we dissected the PROCR signaling pathways in breast cancer cells. Combining protein array, knockdown, and overexpression methods, we found that PROCR concomitantly activates multiple pathways. We also noted that PROCR-dependent ERK and PI3k-Akt-mTOR signaling pathways proceed through Src kinase and transactivation of insulin-like growth factor 1 receptor (IGF-1R). These pathway activities led to the accumulation of c-Myc and cyclin D1. On the other hand, PROCR-dependent RhoA-ROCK-p38 signaling relied on coagulation factor II thrombin receptor (F2R). We confirmed these findings in primary cells isolated from triple-negative breast cancer-derived xenografts (PDX) that have high expression of PROCR. To the best our knowledge, this is the first comprehensive study of PROCR signaling in breast cancer cells, and its findings also shed light on the molecular mechanisms of PROCR in stem cells in normal tissue.

Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells

Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.

Juxtaglomerular cell tumor: Clinical and immunohistochemical features.

Juxtaglomerular cell tumor (JGCT) is a rare tumor, with approximately 100 cases reported in the literature. The authors respectively studied the clinical data of 11 patients diagnosed with JGCT in Peking Union Medical College Hospital from 2004 to 2014, and investigated the immunohistochemical profiles in 10 tumors. Nine of the 11 patients were diagnosed before the age of 40 years. Hypertension was present in all patients, while hypokalemia occurred in seven of 11 patients. Computed tomography detected JGCTs with a sensitivity of 100%. Immunoreactivities for CD34 and vascular endothelial growth factor were observed in most tumor specimens, suggesting that JGCTs express a variety of vessel-related immunohistochemical markers, although JGCTs are considered a tumor without abundant blood supply. Nuclear accumulation of cyclin D1 was common in JGCTs. Results from immunohistochemistry were negative for BRAF, HER2, and TFE3, suggesting that BRAF, HER2, and TFE3 genes might not play a part in tumorigenesis in JGCTs.

Critical roles of Cyclin D1 in mouse embryonic fibroblast cell reprogramming.

Although pluripotent stem cells hold great promise in the fields of human disease modeling and regenerative medicine, the molecular basis of Oct-4, Sox2, Klf4, and c-Myc (OSKM)-induced cellular reprogramming remains unclear. To investigate the molecular mechanisms involved in cellular reprogramming, we studied the immediate effects of expression of the OSKM reprogramming factors on mouse embryonic fibroblasts (MEFs) in this study. Induction of the OSKM reprogramming factors significantly altered primary MEF growth properties. Although MEFs not expressing the reprogramming factors underwent replicative senescence within 9-12 days in culture, MEFs expressing the four reprogramming factors proliferated continuously throughout the duration of the experiment, suggesting that the expression of the OSKM reprogramming factors inhibits or delays replicative senescence. Cell cycle progression by the reprogramming factors was accompanied by the accumulation of Cyclin D1 through the early stages of reprogramming in MEFs, leading us to hypothesize that it might play a positive role in cellular reprogramming. Consistent with this hypothesis, forced Cyclin D1 expression enhanced reprogramming if administered concomitant with expression of the OSKM reprogramming factors. Most importantly, unlike wild-type MEFs expressing reprogramming factors, the number of emerging alkaline phosphatase-positive cyclin D1-null colonies was significantly reduced and cyclin D1-null MEFs were unable to initiate mesenchymal-to-epithelial transition. Our studies demonstrate that cyclin D1 is an essential gene in the reprogramming process and that activation of cyclin D1 by reprogramming factors is an important process for somatic cell reprogramming.

Abstract 3653: Mutational analysis of PARK2, a regulator of cyclin D1 stability, in sporadic parathyroid adenoma

Abstract The molecular mechanisms underlying the pathogenesis of sporadic parathyroid adenomas are incompletely understood. The oncoprotein cyclin D1 is overexpressed in 20-40% of sporadic parathyroid adenomas; however, known aberrations in the gene that encodes cyclin D1, CCND1, account for less than half of these cases. Genetic alterations of PARK2, which encodes the E3 ubiquitin ligase responsible for targeting cyclin D1 for degradation, have been identified as a frequent finding in a variety of human tumors. Interestingly, genetic abnormalities of PARK2 were mutually exclusive of cyclin D1 gene alterations, suggesting a common pathway in tumorigenesis. We postulate that inactivating mutations in PARK2 lead to increased stability of cyclin D1 and thereby may contribute to the development of sporadic parathyroid adenoma. We therefore sought to identify PARK2 coding region mutations in a series of sporadic parathyroid adenomas by direct DNA sequencing. The entire coding region and intron-exon boundaries of PARK2 were examined in a cohort of eighty typically presenting, sporadic parathyroid adenomas. Six known polymorphisms were identified in several of the adenomas in this series; however, none of the polymorphisms are associated with cancer, and no other cancer-associated mutations were found. The absence of intragenic mutations in PARK2 suggests that such mutations do not commonly contribute to the development of sporadic parathyroid adenoma. However, because such intragenic mutations could be rare and limited to only those samples in which overexpression of cyclin D1 cannot be explained by genetic abnormalities in the CCND1 gene, a significant role for cyclin D1 accumulation in parathyroid tumor formation due intragenic inactivating mutations of PARK2 cannot be ruled out. Further research must be conducted in order to elucidate the mechanism(s) of cyclin D1 accumulation in parathyroid adenomas in which CCND1 aberrations are not present. . Citation Format: Kelly Brewer, Jessica Costa-Guda, Andrew Arnold. Mutational analysis of PARK2, a regulator of cyclin D1 stability, in sporadic parathyroid adenoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3653.