Cancer-Testis Antigen MAGE-C2/CT10 Promotes Proliferation and Enhances Resistance to p53 Mediated Apoptosis in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1808-1808
Author(s):  
Nesrine Lajmi ◽  
Julia Templin ◽  
Sara Yousef ◽  
Tim Luetkens ◽  
Stefanie Spock ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Fold Increase ◽  
Myeloma Cell ◽  
Class I ◽  
Cycle Progression ◽  
Myeloma Cells ◽  
Healthy Donors ◽  
Cancer Testis

Abstract Abstract 1808 Background: Cancer-testis antigens belonging to the MAGE class I family of genes are commonly expressed in Multiple Myeloma (MM). Expression of MAGE class I genes is associated with an aggressive clinical course of MM and resistance to chemotherapy, suggesting that MAGE genes may confer a survival advantage on myeloma cells. MAGE-C2/CT10 is member of the MAGE class I family of genes thought to be a good candidate for cancer immunotherapy given its very frequent expression in primary myeloma. In normal cells, MAGE-C2/CT10 seems to suppress p53 expression by promoting its polyubiquitination and degradation. However, the function of MAGE-C2/CT10 in malignancies is completely unknown. We, therefore, investigated for the first time the role of MAGE-C2/CT10 in tumor cells derived from patients with MM. Material and Methods: MAGE-C2/CT10 expression was analysed by real-time PCR and western blot in myeloma cell lines (N=8) and in PBMC from healthy donors (N=8). For the functional evaluation of MAGE-C2/CT10 we decided to use myeloma cell line U-266 which constitutively expresses MAGE-C2/CT10 and a missense mutant p53 (A161T) that has partially lost its transcriptional activity. The biological role of MAGE-C2/CT10 was investigated by stably silencing its expression using lentiviral short hairpin RNA (shRNA). The effects of silencing MAGE-C2/CT10 expression on myeloma cell biology were examined by determining the number of viable or apoptotic cells using a colorimetric MTT assay and annexin-V/7AAD staining followed by flow cytometry, In addition, we measured myeloma cell proliferation and the anchorage-independent growth using a BrdU incorporation assay and a colony formation assay, respectively. Finally, we investigated cell cycle phase distribution by flow cytometry and we analyzed the expression of key molecules involved in cell cycle progression and apoptosis using a real-time PCR array as well as western blot. Results: We found MAGE-C2/CT10 to be constitutively expressed in all myeloma cells lines but not in PBMC from healthy donors. Lentivirus-mediated silencing of MAGE-C2/CT10 inhibited significantly the proliferation and the anchorage-independent growth of myeloma cells. Cell cycle analysis demonstrated that the anti-proliferative effect of MAGE-C2/CT10 silencing in U-266 was due to a 70% decrease of cells in the S phase, a cell cycle arrest at both G0/G1 and G2/M transitions and an increase in the subG0/G1 population due to an activation of apoptotic cell death. The serine-threonine checkpoint effector kinase 2 (CHK2) and its substrate, the tumor suppressor protein p53, are essential for cell cycle control, DNA repair and apoptosis. We found that the loss of MAGE-C2/CT10 expression was associated with the activation of CHK2 through phosphorylation at Thr68 as well as the activation of p53 by phosposphorylation at Ser20. Furthermore, we observed a three-fold increase in the endogenous level of p53 protein which correlated with an up-regulation of two transcriptional targets of p53, the cyclin-dependant kinase inhibitor p21WAF1 and the growth arrest and DNA-damage-inducible alpha protein (GADD45A), known to be essential for p53-induced G1 and G2 arrest, respectively. Finally, using the Human Apoptosis Profiler PCR array that contains a number of p53 target genes, we found that apoptosis induced by MAGE-C2/CT10 knockdown was due to a more than two-fold increase in the transcription of pro-apoptotic genes like BAX (Bcl2-associated × protein), BAD (BCL2-associated agonist of cell death), Cytochrome c, APAF1 (Apoptosis activating factors) as well as several caspases, which are the down-stream mediators of p53-dependant apoptosis in response to DNA damage. Conclusions: Our collected findings support an anti-apoptotic function of MAGE-C2/CT10 in MM, likely through the regulation of key molecules involved in cell cycle progression and p53-mediated apoptosis. The central role of MAGE-C2/CT10 in the biology of myeloma strongly suggest that this cancer-testis antigen represents a promising target for myeloma-specific immunotherapies or other targeted modes of therapy for MM. Disclosures: Kröger: Fresenius Biotech: Honoraria, Research Funding.

β1 Integrin Activation Via FN Adhesion Enhances IL-6 Response in Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2352-2352
Author(s):  
Kenneth H. Shain ◽  
Danielle N. Kelsch ◽  
Mei Huang ◽  
Melissa Alsina ◽  
Richard Jove ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Annexin V ◽  
Fold Increase ◽  
Myeloma Cell ◽  
Cycle Progression ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Stat3 Signaling

Abstract Many environmental factors are known to affect the progression and survival of Multiple Myeloma (MM) cells. These factors include soluble determinants such as cytokines, chemokines and growth factors and physical determinants such as MM cell adhesion to fibronectin (FN) and bone marrow stromal cells. Although these determinants likely work in concert in the in vivo MM microenvironment, they have typically been analyzed individually. Only recently has the idea of crosstalk between different environmental factors been studied and appreciated. In this study we examined signaling events, cell cycle progression, and levels of drug resistance in myeloma cells adhered to FN alone via β1 integrins, stimulated with IL-6 alone, or the two combined. We found that the combination of FN adhesion and IL-6 markedly enhanced myeloma cell Stat3 signaling when compared to either condition alone. Western blot analysis demonstrated that the phosphorylation status of Stat3 was significantly increased by combining IL-6 with FN adhesion as compared to either IL-6 or FN-adhesion alone. Furthermore, Stat3 phosphorylation was sustained with the combination of IL-6 and FN adhesion (up to 6 hours) compared to either treatment alone. A similar effect on Stat3 signaling was observed in U266, H929 and MM1.S myeloma cells. EMSA analysis also demonstrated a synergistic increase in Stat3 nuclear translocation and DNA binding with the combination of IL-6 and FN adhesion, inducing a 3.0-fold increase in protein-DNA complex formation when compared to IL-6 alone and a 10.2-fold increase over FN adhesion alone. Previously, our lab has shown that these factors individually influence cell survival. In this cell line model, the combination of IL-6 and FN adhesion did not confer increased protection to mitoxantrone (a classic topoII inhibitor) beyond that mediated by FN adhesion alone as quantitated by MTT analysis and Annexin V/7-AAD staining (Table 1). We previously demonstrated that IL-6 and FN have differing effects on MM cell proliferation; IL-6 promotes cell cycle progression, whereas FN adhesion facilitates a G0/G1 cell cycle arrest. In the present study, cell cycle analysis (using BrdU/PI) illustrated that the combination of IL-6 and FN adhesion reversed the arrest mediated by FN (Table 1). Together, these data show synergistic effects of IL-6 and FN adhesion on Stat3 signaling that potentially confers a more malignant phenotype. Drug resistance bestowed by adhesion to FN is maintained when myeloma cells are exposed to IL-6, and cells are able to proliferate and overcome the adhesion- mediated cell cycle arrest. Importantly, these results demonstrate that components of the bone marrow microenvironment act synergistically to influence myeloma cell survival and proliferation and understanding how these elements interact provides a new target for myeloma treatment. Table 1 IC50 in nM Percent specific apoptosis Percent G0/G1 IC50 determined by MTT analysis; percent specific apoptosis determined by Annexin V/7-AAD staining; percent G0/G1 determined by BrdU/PI Suspension 20.0+/−2.9 69.9+/−2.2 26.2+/−2.1 Suspension+IL-6 26.5+/−7.5 62.7+/−8.6 24.7+/−4.7 FN 70.8+/−19.9 42.5+/−8.1 35.5+/−3.4 FN+IL-6 66.4+/−18.0 48.2+/−8.5 26.6+/−4.3

Pre-Clinical Evaluation of a Small Molecule Inhibitor of CDK1 as Potential Therapy for MYC Expressing Mutiple Myeloma Tumors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3667-3667 ◽  
Author(s):  
Seda Zeng ◽  
Zhi Hua Li ◽  
Marta Chesi ◽  
Chungyee Leung-Hagesteijn ◽  
Sheng-ben Liang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Myeloma Cell ◽  
S Phase ◽  
Causative Role ◽  
Cyclin D ◽  
Cycle Progression ◽  
Myeloma Cells

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

PTTG has a Dual Role of Promotion-Inhibition in the Development of Pituitary Adenomas

2019 ◽  
Vol 26 (11) ◽  
pp. 800-818
Author(s):  
Zujian Xiong ◽  
Xuejun Li ◽  
Qi Yang
Keyword(s):  
Cell Cycle ◽  
Pituitary Adenoma ◽  
Pituitary Adenomas ◽  
Cell Cycle Progression ◽  
Key Factors ◽  
Cycle Progression ◽  
Sister Chromatids ◽  
Regulation Of Cell Cycle ◽  
Late Stages

Pituitary Tumor Transforming Gene (PTTG) of human is known as a checkpoint gene in the middle and late stages of mitosis, and is also a proto-oncogene that promotes cell cycle progression. In the nucleus, PTTG works as securin in controlling the mid-term segregation of sister chromatids. Overexpression of PTTG, entering the nucleus with the help of PBF in pituitary adenomas, participates in the regulation of cell cycle, interferes with DNA repair, induces genetic instability, transactivates FGF-2 and VEGF and promotes angiogenesis and tumor invasion. Simultaneously, overexpression of PTTG induces tumor cell senescence through the DNA damage pathway, making pituitary adenoma possessing the potential self-limiting ability. To elucidate the mechanism of PTTG in the regulation of pituitary adenomas, we focus on both the positive and negative function of PTTG and find out key factors interacted with PTTG in pituitary adenomas. Furthermore, we discuss other possible mechanisms correlate with PTTG in pituitary adenoma initiation and development and the potential value of PTTG in clinical treatment.

scholarly journals Phosphorylation of RCC1 on Serine 11 Facilitates G1/S Transition in HPV E7-Expressing Cells

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 995
Author(s):  
Xiaoyan Hou ◽  
Lijun Qiao ◽  
Ruijuan Liu ◽  
Xuechao Han ◽  
Weifang Zhang
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Mtor Pathway ◽  
Cycle Progression ◽  
Post Translational Modifications ◽  
Hpv E7 ◽  
Cycle Regulation

Persistent infection of high-risk human papillomavirus (HR-HPV) plays a causal role in cervical cancer. Regulator of chromosome condensation 1 (RCC1) is a critical cell cycle regulator, which undergoes a few post-translational modifications including phosphorylation. Here, we showed that serine 11 (S11) of RCC1 was phosphorylated in HPV E7-expressing cells. However, S11 phosphorylation was not up-regulated by CDK1 in E7-expressing cells; instead, the PI3K/AKT/mTOR pathway promoted S11 phosphorylation. Knockdown of AKT or inhibition of the PI3K/AKT/mTOR pathway down-regulated phosphorylation of RCC1 S11. Furthermore, S11 phosphorylation occurred throughout the cell cycle, and reached its peak during the mitosis phase. Our previous data proved that RCC1 was necessary for the G1/S cell cycle progression, and in the present study we showed that the RCC1 mutant, in which S11 was mutated to alanine (S11A) to mimic non-phosphorylation status, lost the ability to facilitate G1/S transition in E7-expressing cells. Moreover, RCC1 S11 was phosphorylated by the PI3K/AKT/mTOR pathway in HPV-positive cervical cancer SiHa and HeLa cells. We conclude that S11 of RCC1 is phosphorylated by the PI3K/AKT/mTOR pathway and phosphorylation of RCC1 S11 facilitates the abrogation of G1 checkpoint in HPV E7-expressing cells. In short, our study explores a new role of RCC1 S11 phosphorylation in cell cycle regulation.

scholarly journals EGFR-ERK induced activation of GRHL1 promotes cell cycle progression by up-regulating cell cycle related genes in lung cancer

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Yiming He ◽  
Mingxi Gan ◽  
Yanan Wang ◽  
Tong Huang ◽  
Jianbin Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Potential Role ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Phosphorylation Site ◽  
Promoter Regions ◽  
Cycle Progression

AbstractGrainyhead-like 1 (GRHL1) is a transcription factor involved in embryonic development. However, little is known about the biological functions of GRHL1 in cancer. In this study, we found that GRHL1 was upregulated in non-small cell lung cancer (NSCLC) and correlated with poor survival of patients. GRHL1 overexpression promoted the proliferation of NSCLC cells and knocking down GRHL1 inhibited the proliferation. RNA sequencing showed that a series of cell cycle-related genes were altered when knocking down GRHL1. We further demonstrated that GRHL1 could regulate the expression of cell cycle-related genes by binding to the promoter regions and increasing the transcription of the target genes. Besides, we also found that EGF stimulation could activate GRHL1 and promoted its nuclear translocation. We identified the key phosphorylation site at Ser76 on GRHL1 that is regulated by the EGFR-ERK axis. Taken together, these findings elucidate a new function of GRHL1 on regulating the cell cycle progression and point out the potential role of GRHL1 as a drug target in NSCLC.

Mutations at sites involved in Suc1 binding inactivate Cdc2

1991 ◽  
Vol 11 (12) ◽  
pp. 6177-6184
Author(s):  
B Ducommun ◽  
P Brambilla ◽  
G Draetta
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Physiological Role ◽  
Negative Effects ◽  
Alanine Scanning Mutagenesis ◽  
Cycle Progression ◽  
Mutant Proteins ◽  
Additional Protein

suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function.

scholarly journals Evidence for a role of spindle matrix formation in cell cycle progression by antibody perturbation

PLoS ONE ◽  
2018 ◽  
Vol 13 (11) ◽  
pp. e0208022 ◽  
Author(s):  
Changfu Yao ◽  
Chao Wang ◽  
Yeran Li ◽  
Michael Zavortink ◽  
Vincent Archambault ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Spindle Matrix

scholarly journals Role of AKT/mTORC1 pathway in pancreatic β-cell proliferation

2012 ◽  
pp. 235-243 ◽  
Author(s):  
Norman Balcazar Morales ◽  
Cecilia Aguilar de Plata
Keyword(s):  
Cell Cycle ◽  
Growth Factors ◽  
Cell Cycle Progression ◽  
Cell Mass ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Cycle Progression ◽  
Mtorc1 Signaling

Growth factors, insulin signaling and nutrients are important regulators of β-cell mass and function. The events linking these signals to regulation of β-cell mass are not completely understood. Recent findings indicate that mTOR pathway integrates signals from growth factors and nutrients with transcription, translation, cell size, cytoskeleton remodeling and mitochondrial metabolism. mTOR is a part of two distinct complexes; mTORC1 and mTORC2. The mammalian TORC1 is sensitive to rapamycin and contains Raptor, deptor, PRAS40 and the G protein β-subunit-like protein (GβL). mTORC1 activates key regulators of protein translation; ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1. This review summarizes current findings about the role of AKT/mTORC1 signaling in regulation of pancreatic β cell mass and proliferation. mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclins D2, D3 and cdk4/cyclin D activity. These studies uncovered key novel pathways controlling cell cycle progression in β-cells in vivo. This information can be used to develop alternative approaches to expand β-cell mass in vivo and in vitro without the risk of oncogenic transformation. The acquisition of such knowledge is critical for the design of improved therapeutic strategies for the treatment and cure of diabetes as well as to understand the effects of mTOR inhibitors in β-cell function.

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