Inhibition of REV3 Expression Induces Persistent DNA Damage and Growth Arrest in Cancer Cells
TL;DR: The findings indicate that depletion of REV3 not only can amend cisplatin-based cancer therapy but also can be applied for susceptible cancers as a potential monotherapy.
About: This article is published in Neoplasia.The article was published on 2011-10-01 and is currently open access. It has received 53 citations till now. The article focuses on the topics: Cancer cell & Cell aging.
Citations
More filters
TL;DR: Using a large dataset of CRC miRNA and gene expression profiles, the interplay of miRNA groups in regulating gene expression is described, which in turn affects modulated pathways that are important for tumor development.
Abstract: Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed. Most changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples. Using a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development.
145 citations
TL;DR: In normal cells, serum starvation in vitro induces a cell cycle arrest and protects from CDDP induced toxicity and has the potential to enhance the therapeutic index of cisplatin-based therapy.
Abstract: Optimizing the safety and efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is of clinical relevance. Serum starvation in vitro and short-term food starvation in vivo both stress cells by the sudden depletion of paracrine growth stimulation. The effects of serum starvation on CDDP toxicity were investigated in normal and cancer cells by assessing proliferation, cell cycle distribution and activation of DNA-damage response and of AMPK, and were compared to effects observed in cells grown in serum-containing medium. The effects of short-term food starvation on CDDP chemotherapy were assessed in xenografts-bearing mice and were compared to effects on tumor growth and/or regression determined in mice with no diet alteration. We observed that serum starvation in vitro sensitizes cancer cells to CDDP while protecting normal cells. In detail, in normal cells, serum starvation resulted in a complete arrest of cellular proliferation, i.e. depletion of BrdU-incorporation during S-phase and accumulation of the cells in the G0/G1-phase of the cell cycle. Further analysis revealed that proliferation arrest in normal cells is due to p53/p21 activation, which is AMPK-dependent and ATM-independent. In cancer cells, serum starvation also decreased the fraction of S-phase cells but to a minor extent. In contrast to normal cells, serum starvation-induced p53 activation in cancer cells is both AMPK- and ATM-dependent. Combination of CDDP with serum starvation in vitro increased the activation of ATM/Chk2/p53 signaling pathway compared to either treatment alone resulting in an enhanced sensitization of cancer cells to CDDP. Finally, short-term food starvation dramatically increased the sensitivity of human tumor xenografts to cisplatin as indicated not only by a significant growth delay, but also by the induction of complete remission in 60% of the animals bearing mesothelioma xenografts, and in 40% of the animals with lung carcinoma xenografts. In normal cells, serum starvation in vitro induces a cell cycle arrest and protects from CDDP induced toxicity. In contrast, proliferation of cancer cells is only moderately reduced by serum starvation whereas CDDP toxicity is enhanced. The combination of CDDP treatment with short term food starvation improved outcome in vivo. Therefore, starvation has the potential to enhance the therapeutic index of cisplatin-based therapy.
108 citations
Cites methods from "Inhibition of REV3 Expression Induc..."
...2 (p53−/−) [17], and primary normal human mesothelial SDM104 [40] cells were cultured in M199 (Invitrogen)/MCDB106 (Sigma) (1:1) mixed medium supplemented with 15% FCS, 10 ng/ml EGF, and 0....
[...]
...Human mesothelioma ZL55 [38], lung carcinoma A549 [39] cells, the colorectal carcinoma cell lines HCT116 40.16 (p53+/+) and HCT116 379.2 (p53−/−) [17], and primary normal human mesothelial SDM104 [40] cells were cultured in M199 (Invitrogen)/MCDB106 (Sigma) (1:1) mixed medium supplemented with 15% FCS, 10 ng/ml EGF, and 0.4μg/ml hydrocortisone as described [41]....
[...]
...C, the quantification of flow cytometry results of SDM104 alone, or both together is shown (n=3)....
[...]
...Anti-phosphoATM-Ser1981 (pATM) immuno-staining of untreated SDM104 cells (A) and those treated with 8 μM CDDP alone (B), serum starvation alone (C), or both together (D) are shown....
[...]
...MTT assays were performed after primary normal cell cultures LP9, SDM104 and SDM85, which was established from a normal pleural tissue received from a patient undergoing cancer unrelated thoracic surgery (this study was approved by the Zurich University Hospital ethic committee and a written informed consent was obtained from the patient), were treated with CDDP alone, serum starvation alone or both together (* for P<0.002; ** for P<3.0x10-5)....
[...]
TL;DR: The first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin and it is reported that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds.
Abstract: The DNA damage response kinase ATR may be a useful cancer therapeutic target ATR inhibition synergizes with loss of ERCC1, ATM, XRCC1 and DNA damaging chemotherapy agents Clinical trials have begun using ATR inhibitors in combination with cisplatin Here we report the first synthetic lethality screen with a combination treatment of an ATR inhibitor (ATRi) and cisplatin Combination treatment with ATRi/cisplatin is synthetically lethal with loss of the TLS polymerase ζ and 53BP1 Other DNA repair pathways including homologous recombination and mismatch repair do not exhibit synthetic lethal interactions with ATRi/cisplatin, even though loss of some of these repair pathways sensitizes cells to cisplatin as a single-agent We also report that ATRi strongly synergizes with PARP inhibition, even in homologous recombination-proficient backgrounds Lastly, ATR inhibitors were able to resensitize cisplatin-resistant cell lines to cisplatin These data provide a comprehensive analysis of DNA repair pathways that exhibit synthetic lethality with ATR inhibitors when combined with cisplatin chemotherapy, and will help guide patient selection strategies as ATR inhibitors progress into the cancer clinic
101 citations
Cites background from "Inhibition of REV3 Expression Induc..."
...Loss of REV3 also causes persistent DNA damage and an increase in γH2AX [48]....
[...]
...53BP1-depleted cells also showed an increase in γH2AX staining when treated with the ATR inhibitor, indicative of DNA damage (Fig 9B and 9C)....
[...]
...Cells were then fixed and stained with DAPI to visualize nuclei (A) and γH2AX to identify sites of DNA damage (B)....
[...]
...(C) Quantification of the γH2AX intensity of cells shown in B. Box and whiskers plot shows the mean and the range of the samples, * p<0.01, ns (not significant). doi:10.1371/journal.pone.0125482.g009 PLOS ONE | DOI:10.1371/journal.pone.0125482 May 12, 2015 16 / 22 53BP1 is also required to protect under-replicated regions of the genome, such as common fragile sites, during mitosis for repair in the subsequent G1....
[...]
TL;DR: The observations that GAS5 levels modify cell proliferation in vitro, and that G AS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS 5 in MPm biology.
Abstract: Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA “sponge”. Our aim was to investigate the possible role of the GAS5 in the growth of MPM. Primary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry. GAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression. The observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.
74 citations
TL;DR: The specialized functions of Y family polymerases (Rev1, Polη, Polι and Polκ) and DNA polymerase ζ in lesion bypass, mutagenesis, and prevention of genome instability are described, the latter due to newly appreciated roles in DNA repair.
Abstract: Cancer cells display numerous abnormal characteristics which are initiated and maintained by elevated mutation rates and genome instability. Chromosomal DNA is continuously surveyed for the presence of damage or blocked replication forks by the DNA Damage Response (DDR) network. The DDR is complex and includes activation of cell cycle checkpoints, DNA repair, gene transcription, and induction of apoptosis. Duplicating a damaged genome is associated with elevated risks to fork collapse and genome instability. Therefore, the DNA damage tolerance (DDT) pathway is also employed to enhance survival and involves the recruitment of translesion DNA synthesis (TLS) polymerases to sites of replication fork blockade or single stranded DNA gaps left after the completion of replication in order to restore DNA to its double stranded form before mitosis. TLS polymerases are specialized for inserting nucleotides opposite DNA adducts, abasic sites, or DNA crosslinks. By definition, the DDT pathway is not involved in the actual repair of damaged DNA, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and lessening the chance for genome instability. However this may be associated with increased mutagenesis. In this review, we will describe the specialized functions of Y family polymerases (Rev1, Polη, Polι and Polκ) and DNA polymerase ζ in lesion bypass, mutagenesis, and prevention of genome instability, the latter due to newly appreciated roles in DNA repair. The recently described role of the Fanconi anemia pathway in regulating Rev1 and Polζ-dependent TLS is also discussed in terms of their involvement in TLS, interstrand crosslink repair, and homologous recombination.
72 citations
Cites background from "Inhibition of REV3 Expression Induc..."
...If it holds true, that cancer cells are more dependent upon Polζ for growth and survival (and perhaps HR repair) as suggested by Knobel et al [243], we anticipate that a potential new Achilles heal may have been identified....
[...]
...Large changes in expression levels may be incompatible with cell survival and hence selected against [242, 243]....
[...]
References
More filters
TL;DR: It is shown that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture, which provides in situ evidence that senescent cells may exist and accumulate with age in vivo.
Abstract: Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.
6,696 citations
TL;DR: The survival curves obtained with human diploid cell strains are comparable to “multiple-hit” or “ multiple-target” curves obtain with other biological systems where an initial threshold dose is required before an exponential form of the curve is established.
5,562 citations
TL;DR: It is shown that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions commonly express markers of an activated DNA damage response.
Abstract: During the evolution of cancer, the incipient tumour experiences 'oncogenic stress', which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM-Chk2-p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.
2,641 citations
TL;DR: In addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.
Abstract: Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Furthermore, DDR activity and IL-6 were elevated in human cancers, and ATM-depletion suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.
1,793 citations
TL;DR: Evidence is presented for a large class of non-oncogenes that are essential for cancer cell survival and present attractive drug targets and theoretical considerations for combining orthogonal cancer therapies are provided.
1,619 citations