Prognostic and therapeutic significance of ribonucleotide reductase small subunit M2 in prostate cancer.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 240-240
Author(s):  
Ying Zhang Mazzu ◽  
Travis A. Gerke ◽  
Goutam Chakraborty ◽  
Joshua Armenia ◽  
Mohammad Omar Atiq ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Growth ◽  
Protein Expression ◽  
Ribonucleotide Reductase ◽  
Rna Seq ◽  
Oncogenic Signaling ◽  
Downstream Targets

240 Background: DNA-repair defects are common in advanced prostate cancer (PC) and high-risk localized tumors. Besides deficiency of DNA repair, overexpression of DNA repair genes could also contribute to poorer outcomes for PC patients. The nucleotide metabolism enzyme ribonucleotide reductase (RNR) plays the key role in DNA synthesis and repair. RRM2, the rate-limiting RNR subunit, is frequently up-regulated in cancer, and its overexpression leads to chemoresistance. Although targeting RRM2 by siRNA and small molecules has been applied in clinical trials in multiple cancers, limited knowledge of RRM2 function in PC delays potential clinical application of RRM2 inhibition. Methods: We leveraged publically available PC clinical cohorts to examine RRM2 levels and clinical outcomes. siRNAs was applied to knockdown of RRM2 in multiple PC cell lines. Cell growth, cell cycle and apoptosis were analyzed to determine siRRM2 or RRM2 inhibitor (COH29)-induced phenotypes. RNA-seq and protein array were performed to identify downstream targets of RRM2. Immunohistochemistry staining was applied to determine prevalence of RRM2 protein expression in PC tissues microarrays (TMAs). Results: In PC cohorts, increased RRM2 expression was associated with a higher likelihood of metastasis, poorer disease-free survival, and increased risk of development of lethal disease (N = 1200, PHS/HPFS cohorts). In PC cells, Inhibition of RRM2 induced remarkable cell growth inhibition, cell cycle arrest (at S phase) and apoptosis. DNA damage was observed in siRRM2/COH29-treated PC cells with increased activation of DNA damage markers. GSEA analysis of the RNA-seq dataset revealed multiple biological processes were affected by inhibition of RRM2, such as cell cycle, apoptosis, and DNA damage response. Intriguingly, MYC oncogenic signaling is the major downstream targets of RRM2. Furthermore, inhibition of RRM2 can block multiple oncogenic signaling including mTOR/AKT, SFK, and STAT signaling by repressing the key phospho-kinases in PC cells. Among 121 cases on the PC TMAs, 20% showed strong RRM2 protein expression. Conclusions: RRM2 may serve as a prognostic biomarker and novel therapeutic target in PC.

Differential response of prostate cancer cells to ionizing radiation: The RB status.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 106-106
Author(s):  
Robert Benjamin Den ◽  
Steve Ciment ◽  
Ankur Sharma ◽  
Hestia Mellert ◽  
Steven McMahon ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Ionizing Radiation ◽  
Cell Survival ◽  
Hormone Sensitive ◽  
Clonogenic Cell ◽  
Castrate Resistant

106 Background: Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer death in U.S. men. The retinoblastoma tumor suppressor protein, RB, plays a critical role in cell cycle regulation and loss of RB has been observed in 25-30% of prostate cancers. We have previously shown that RB loss results in a castrate resistant phenotype, however the consequences of RB status with regard to radiation response are unknown. We hypothesized that RB loss would downregulate the G1-S cell cycle checkpoint arrest normally induced by irradiation, inhibit DNA repair, and subsequently sensitize cells to ionizing radiation. Methods: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAP-C4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of RB using shRNA). Gamma H2AX assays were used to quantitate DNA damage and PARP cleavage and Caspase 3 were used to quantitate apoptosis. FACS analysis with BrdU was used to assess the cell cycle. Cell survival was measured using the clonogenic cell survival assay. In vivo work was performed in nude mice with tumor xenografts. Results: We observed that loss of RB increased radioresponsiveness in both transient and clonogenic cell survival assays in both hormone sensitive and castrate resistant cell lines (p<0.05). Cell death was not mediated through increased apoptosis nor was perturbations in cell cycle noted. However, loss of RB effected DNA repair as measured by gamma H2AX staining as well as cellular senescence. In vivo xenografts of the RB deficient tumors exhibited diminished tumor mass, lower PSA kinetics and decreased tumor growth after treatment with single fraction of ionizing radiation in comparison to RB intact tumors (p<0.05). Conclusions: Loss of RB results in a differential response to ionizing radiation. Isogenic cells with RB knockdown are more sensitive to DNA damage and result in reduced cell survival. The underlying mechanism appears to be related to DNA damage repair and cellular senescence.

scholarly journals NU7441 Enhances the Radiosensitivity of Liver Cancer Cells

2016 ◽  
Vol 38 (5) ◽  
pp. 1897-1905 ◽  
Author(s):  
Chuanjie Yang ◽  
Quanxu Wang ◽  
Xiaodan Liu ◽  
Xiulian Cheng ◽  
Xiaoyu Jiang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Liver Cancer ◽  
Protein Expression ◽  
Cancer Cells ◽  
Hepg2 Cells ◽  
Specific Inhibitor ◽  
Cell Counting ◽  
Liver Cancer Cells

Objective: Radiation therapy, one of the major treatments for liver cancer, causes DNA damage and cell death. Since the liver cancer cells have a strong capacity to repair irradiative injury, new medicines to enhance this treatment are urgently required. In this study, we investigated the effect of NU7441, a synthetic small-molecule compound, as a specific inhibitor of DNA-dependent protein kinase (DNA-PK) in radiosensitization of hepatocellular carcinoma HepG2 cells. Methods: Cell Counting Kit-8 (CCK-8) was first used to evaluate the proliferation of HepG2 cells under NU7441 treatment. SDS-PAGE and Western blot were then performed to study the protein expression leading to the DNA damage repair. Further, neutral single cell gel electrophoresis and immunofluorescence assay were carried out to assess DNA repair. Finally, flow cytometry was implemented to examine the changes in cell cycle. Results: NU7441 reduced the CCK-8 counts in the HepG2 culture, further enhanced 60Coγ radiation injury to HepG2 cells, which was manifested by decreasing the DNA-PKcs (S2056) protein expression, increasing γH2AX foci number, prolonging the tail moment of the comet cells, and inducing cell cycle arrest at G2/M phase. Conclusion: NU7441 inhibited the growth of liver cancer cells, enhanced the radiosensitization of these cancer cells by interfering with the DNA repair and cell cycle checkpoint. These data implicate NU7441 as a potential radiotherapy sensitizer for the treatment of liver cancer.

scholarly journals Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Zachary L. Watson ◽  
Tomomi M. Yamamoto ◽  
Alexandra McMellen ◽  
Hyunmin Kim ◽  
Connor J. Hughes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Ovarian Carcinoma ◽  
Immunofluorescent Staining ◽  
High Grade ◽  
Rna Seq ◽  
Serous Ovarian Carcinoma ◽  
Resistant Cells

Abstract Background Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown. Results To identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR. Conclusions This study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.

scholarly journals Inhibition of PIM Kinases Mitigates DNA Repair Responses Following Anthracycline-Induced DNA Damage and Enhances the Anti-Tumor Activity of Doxorubicin Against Lymphoma Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4505-4505 ◽  
Author(s):  
Jeffrey D. Altenburg ◽  
Shuhong Zhang ◽  
Michelle Grimard ◽  
Xingkui Xue ◽  
Sherif S Farag
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Cell Growth ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Fold Increase ◽  
Pim Kinases ◽  
Anti Cancer

Abstract The PIM kinases are a family of proteins recently identified as promising therapeutic targets in several cancers, including pancreatic, B-cell malignancies, acute leukemia, and prostate cancer among others. The family of PIM kinases is composed of three different members (PIM-1, -2, and -3) that are short-lived serine/threonine kinases involved in the regulation of a number of cellular pathways that are important for cancer cell growth and survival. The PIM kinases show high homology with each other, and exhibit functional redundancy in vitro and in vivo. Overexpression of PIM kinases promotes tumor growth through activation of several key cell-cycle progression and anti-apoptotic proteins, including BAD, p21, p27KIP, c-Myc, and AKT-1. Recently, overexpression of PIM-2 has been shown to have a protective effect against ultraviolet light induced DNA damage (Zirkin et al. J Biol Chem288:21770-83, 2013). We investigated the protective role of PIM kinases in chemotherapy-induced DNA damage, and whether inhibition of PIM kinases enhances anthracycline-induced DNA damage by inhibiting DNA repair, thus enhancing cell death in lymphoma cells lines. Using immunobloting and RT-PCR, we found similarly low levels of PIM-1 and PIM-3, but a wide range of PIM-2 expression, in a panel of non-Hodgkin lymphoma (NHL) cell lines, including Raji, HS Sultan, Daudi, Farage, Granta519, and Toledo. Treatment of cells with doxorubicin (200-400 nM) resulted in up to a five fold increase gene transcription and expression of PIM-1 and PIM-2, which was maximal at 6 hours, and was associated with an increase in DNA damage as detected using acridine orange flow cytometry assay. We also tested the single agent effect of the pan-PIM kinase inhibitor, CX6258 on the cell lines. CX6258 alone inhibited cell growth in all NHL cell lines with varying degrees of potency with IC50ranging from 0.2 – 12.9 µM. The anti-cancer was associated most with PIM-2 expression, with the most sensitive cell lines, Daudi and Toledo, expressing the most PIM-2. Suppression of PIM-2 expression by shRNA significantly decreased proliferation, indicating that PIM-2 is a significant factor in cell growth. Treatment of NHL cells with CX6258 resulted in increased caspase-3 activation and PARP cleavage, decreased BAD phosphorylation, and apoptosis. Treatment with CX6258 also increased expression of p21, decreased expression of cyclins A1 and B1, and induced G2-M cell cycle arrest. The effect of combinations of CX6258 (5-50 µM) and doxorubicin (50-500 nM) on DNA damage and cell death was tested on HS sultan and Daudi cells. While doxorubicin alone resulted in a two-fold increase in DNA damage, this was significantly increased in the presence of CX6258 (12 fold). The addition of CX6258 inhibited the phosphorylation of the DNA repair proteins H2.AX, ATM, and Chk2 that occurred when the cells were treated with doxorubicin alone. The combination of CX6258 and doxorubicin was synergistic in inducing lymphoma cell death, with combination indexes ranging from 0.32-0.85. Our findings suggest a mechanism for synergy where doxorubicin damages cellular DNA and initiates the DNA damage response, while CX6258 inhibits the upregulated PIM kinases from activating the proteins involved in the response. This synergistic anti-tumor activity is further strengthened by the CX6258 inhibition of cell cycle progression and anti-apoptotic proteins activated by the PIM kinases. Taken together, our results provide pre-clinical rationale for clinical testing of PIM kinase inhibitors in combination with doxorubicin in patients with NHL. It also suggests that CX6258 may similarly enhance the anti-cancer effects of other DNA damaging agents. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Interactions of DNA Repair, Telomere Homeostasis, and p53 Mutational Status in Solid Cancers: Risk, Prognosis, and Prediction

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 479
Author(s):  
Pavel Vodicka ◽  
Ladislav Andera ◽  
Alena Opattova ◽  
Ludmila Vodickova
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Lesions ◽  
Cell Cycle Checkpoint ◽  
Solid Cancer ◽  
Genomic Integrity ◽  
Repair Capacity ◽  
Mutational Status ◽  
Telomere Homeostasis

The disruption of genomic integrity due to the accumulation of various kinds of DNA damage, deficient DNA repair capacity, and telomere shortening constitute the hallmarks of malignant diseases. DNA damage response (DDR) is a signaling network to process DNA damage with importance for both cancer development and chemotherapy outcome. DDR represents the complex events that detect DNA lesions and activate signaling networks (cell cycle checkpoint induction, DNA repair, and induction of cell death). TP53, the guardian of the genome, governs the cell response, resulting in cell cycle arrest, DNA damage repair, apoptosis, and senescence. The mutational status of TP53 has an impact on DDR, and somatic mutations in this gene represent one of the critical events in human carcinogenesis. Telomere dysfunction in cells that lack p53-mediated surveillance of genomic integrity along with the involvement of DNA repair in telomeric DNA regions leads to genomic instability. While the role of individual players (DDR, telomere homeostasis, and TP53) in human cancers has attracted attention for some time, there is insufficient understanding of the interactions between these pathways. Since solid cancer is a complex and multifactorial disease with considerable inter- and intra-tumor heterogeneity, we mainly dedicated this review to the interactions of DNA repair, telomere homeostasis, and TP53 mutational status, in relation to (a) cancer risk, (b) cancer progression, and (c) cancer therapy.

scholarly journals Dehydroepiandrosterone Administration or Gαq Overexpression Induces β-Catenin/T-Cell Factor Signaling and Growth via Increasing Association of Estrogen Receptor-β/Dishevelled2 in Androgen-Independent Prostate Cancer Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1428-1440 ◽  
Author(s):  
Xunxian Liu ◽  
Julia T. Arnold ◽  
Marc R. Blackman
Keyword(s):  
Prostate Cancer ◽  
Estrogen Receptor ◽  
T Cell ◽  
Cell Growth ◽  
Protein Expression ◽  
Cyclin D1 ◽  
D1 Protein ◽  
Nutritional Supplement ◽  
T Cell Factor ◽  
Androgen Independent

β-Catenin/T-cell factor signaling (β-CTS) plays multiple critical roles in carcinogenesis and is blocked by androgens in androgen receptor (AR)-responsive prostate cancer (PrCa) cells, primarily via AR sequestration of β-catenin from T-cell factor. Dehydroepiandrosterone (DHEA), often used as an over-the-counter nutritional supplement, is metabolized to androgens and estrogens in humans. The efficacy and safety of unregulated use of DHEA are unclear. We now report that DHEA induces β-CTS via increasing association of estrogen receptor (ER)-β with Dishevelled2 (Dvl2) in AR nonresponsive human PrCa DU145 cells, a line of androgen-independent PrCa (AiPC) cells. The induction is temporal, as assessed by measuring kinetics of the association of ERβ/Dvl2, protein expression of the β-CTS targeted genes, c-Myc and cyclin D1, and cell growth. However, in PC-3 cells, another human AiPC cell line, DHEA exerts no detectible effects, partly due to their lower expression of Gα-subunits and DHEA down-regulation of ERβ/Dvl2 association. When Gαq is overexpressed in PC-3 cells, β-CTS is constitutively induced, including increasing c-Myc and cyclin D1 protein expression. This effect involved increasing associations of Gαq/Dvl2 and ERβ/Dvl2 and promoted cell growth. These activities require ERβ in DU-145 and PC-3 cells because they are blocked by ICI 182–780 treatment inactivating ERβ, small interfering RNA administration depleting ERβ, or AR overexpression arresting ERβ. These data suggest that novel pathways activating β-CTS play roles in the progression of AiPC. Although DHEA may enhance PrCa cell growth via androgenic or estrogenic pathways, the effects of DHEA administration on clinical prostate function remain to be determined.

Gallic acid provokes DNA damage and suppresses DNA repair gene expression in human prostate cancer PC-3 cells

2011 ◽  
Vol 28 (10) ◽  
pp. 579-587 ◽  
Author(s):  
Kuo-Ching Liu ◽  
Heng-Chien Ho ◽  
An-Cheng Huang ◽  
Bin-Chuan Ji ◽  
Hui-Yi Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Dna Damage ◽  
Dna Repair ◽  
Gallic Acid ◽  
Human Prostate ◽  
Human Prostate Cancer ◽  
Repair Gene ◽  
Dna Repair Gene

scholarly journals Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer

2020 ◽  
Vol 295 (50) ◽  
pp. 17169-17186
Author(s):  
Mysore S. Veena ◽  
Santanu Raychaudhuri ◽  
Saroj K. Basak ◽  
Natarajan Venkatesan ◽  
Parameet Kumar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Dna Damage ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Cell ◽  
Dna Damage Response ◽  
S Phase ◽  
Cervical Cancer Cell ◽  
Damage Response

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

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