TP53 regulates miRNA association with AGO2 to remodel the miRNA-mRNA interaction network.

DNA damage activates TP53-regulated surveillance mechanisms that are crucial in suppressing tumorigenesis. TP53 orchestrates these responses directly by transcriptionally modulating genes, including microRNAs (miRNAs), and by regulating miRNA biogenesis through interacting with the DROSHA complex. However, whether the association between miRNAs and AGO2 is regulated following DNA damage is not yet known. Here, we show that, following DNA damage, TP53 interacts with AGO2 to induce or reduce AGO2's association of a subset of miRNAs, including multiple let-7 family members. Furthermore, we show that specific mutations in TP53 decrease rather than increase the association of let-7 family miRNAs, reducing their activity without preventing TP53 from interacting with AGO2. This is consistent with the oncogenic properties of these mutants. Using AGO2 RIP-seq and PAR-CLIP-seq, we show that the DNA damage-induced increase in binding of let-7 family members to the RISC complex is functional. We unambiguously determine the global miRNA-mRNA interaction networks involved in the DNA damage response, validating them through the identification of miRNA-target chimeras formed by endogenous ligation reactions. We find that the target complementary region of the let-7 seed tends to have highly fixed positions and more variable ones. Additionally, we observe that miRNAs, whose cellular abundance or differential association with AGO2 is regulated by TP53, are involved in an intricate network of regulatory feedback and feedforward circuits. TP53-mediated regulation of AGO2-miRNA interaction represents a new mechanism of miRNA regulation in carcinogenesis.

The role of TP53 in miRNA loading onto AGO2 and in remodelling the miRNA-mRNA interaction network.

BACKGROUND: DNA damage transactivates tumour protein p53 (TP53)-regulated surveillance, crucial in suppressing tumorigenesis. TP53 mediates this process directly by transcriptionally modulating gene and microRNA (miRNA) expression and indirectly by regulating miRNA biogenesis. However, the role of TP53 in regulating miRNA-AGO2 loading and global changes in AGO2 binding to its gene targets in response to DNA damage are unknown. These processes might be novel mechanisms by which TP53 regulates miRNAs in response to DNA damage. METHODS: To show the network of miRNA-mRNA interactions that occur in response to DNA damage, we stimulated TP53 wild-type and null cell-lines with doxorubicin and performed RNA sequencing from total RNA (RNA-Seq) and AGO2-immunoprecipitated RNA (AGO2-RIP-Seq). We used a combined AGO2 RIP-seq and AGO2 PAR-CLIP-seq (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) approach to determine the exact sites of interaction between the AGO2-bound miRNAs and their mRNA targets. FINDINGS: TP53 directly associated with AGO2, and induced and reduced loading of a subset of miRNAs, including the lethal 7 (let-7) miRNA family members, onto AGO2 in response to DNA damage. Although mutated TP53 maintained its capacity to interact with AGO2, it mediated unloading instead of loading of let-7 family miRNAs, thereby reducing their activity. We determined the miRNA-mRNA interaction networks involved in the response to DNA damage both in the presence and absence of TP53. Furthermore, we showed that miRNAs whose cellular abundance or differential loading onto AGO2 was regulated by TP53 were involved in an intricate network of regulatory feedback and feedforward circuits that fine-tuned gene expression levels in response to DNA damage to permit the repair of DNA damage or initiation of programmed cell death. INTERPRETATION: Control of AGO2 loading by TP53 is a new mechanism of miRNA regulation in carcinogenesis. FUNDING: UK Medical Research Council, Action Against Cancer.

The efficacy of irinotecan, paclitaxel, and oxaliplatin (IPO) in relapsed germ cell tumours with high-dose chemotherapy as consolidation: a non-cisplatin-based induction approach.

OBJECTIVES: To determine the outcome of an expanded cohort of patients with relapsed germ cell tumours (GCTs) treated with a salvage chemotherapy regimen consisting of irinotecan, paclitaxel and oxaliplatin (IPO) and assess the role of IPO as an alternative to standard cisplatin-based chemotherapy regimens in this setting. PATIENTS AND METHODS: The results of 72 consecutive patients were reviewed retrospectively. IPO was used either as a second-line treatment (29 patients), of which 20 patients subsequently received high-dose chemotherapy (HDCT), or third-line (43), of which 32 patients proceeded to HDCT. RESULTS: The 2-year progression-free survival (PFS) and 3-year overall survival (OS) rates for the whole cohort were 30.2% (95% confidence interval [CI] 17.3-40.5%) and 33.4% (95% CI 20.1-43.8%), respectively. Complete remission was achieved in 3%, marker-negative partial response (PR) in 41%, marker-positive PR in 18%, stable disease in 17% and progressive disease in 20%. In the second-line setting, the 2-year PFS rate was 43.5% (95% CI 21.7-60.8%) and 3-year OS 49.1% (95% CI 24.2-65.1%). In the third-line setting, the 2-year PFS rate was 21.0% (95% CI 9.5-35.4%) and the 3-year OS rate was 23.9% (95% CI 11.7-38.2). According to the current international prognostic factor study group criteria for first relapse for the high- and very high-risk group the 2-year PFS rates were 50% and 30%, respectively. There were two treatment-related deaths from IPO, and four from HDCT. Grade 3 or 4 toxicities included neutropenia (35%), thrombocytopaenia (18%), infection (15%), diarrhoea (11%) and lethargy (8%). CONCLUSIONS: IPO offers an effective, well-tolerated, non-nephrotoxic alternative to cisplatin-based salvage regimens for patients with relapsed GCTs. It appears particularly useful in high-risk patients and for those in whom cisplatin is ineffective or contra-indicated.

MicroRNAs cooperatively inhibit a network of tumor suppressor genes to promote pancreatic tumor growth and progression.

BACKGROUND & AIMS: There has not been a broad analysis of the combined effects of altered activities of microRNAs (miRNAs) in pancreatic ductal adenocarcinoma (PDAC) cells, and it is unclear how these might affect tumor progression or patient outcomes. METHODS: We combined data from miRNA and messenger RNA (mRNA) expression profiles and bioinformatic analyses to identify an miRNA-mRNA regulatory network in PDAC cell lines (PANC-1 and MIA PaCa-2) and in PDAC samples from patients. We used this information to identify miRNAs that contribute most to tumorigenesis. RESULTS: We identified 3 miRNAs (MIR21, MIR23A, and MIR27A) that acted as cooperative repressors of a network of tumor suppressor genes that included PDCD4, BTG2, and NEDD4L. Inhibition of MIR21, MIR23A, and MIR27A had synergistic effects in reducing proliferation of PDAC cells in culture and growth of xenograft tumors in mice. The level of inhibition was greater than that of inhibition of MIR21 alone. In 91 PDAC samples from patients, high levels of a combination of MIR21, MIR23A, and MIR27A were associated with shorter survival times after surgical resection. CONCLUSIONS: In an integrated data analysis, we identified functional miRNA-mRNA interactions that contribute to growth of PDACs. These findings indicate that miRNAs act together to promote tumor progression; therapeutic strategies might require inhibition of several miRNAs.

Brain metastases associated with germ cell tumors may be treated with chemotherapy alone.

BACKGROUND: The management of brain metastases in patients with germ cell tumors remains controversial. The authors assessed the outcome in this patient group after the introduction of GAMEC chemotherapy (14-day cisplatin, high-dose methotrexate, etoposide, and actinomycin-D with filgrastim support) and cessation of the routine use of cranial irradiation. METHODS: Data were recorded prospectively from 39 patients with germ cell tumors and concurrent brain metastases who received treatment before and after the advent of GAMEC after they relapsed on conventional cisplatin-based chemotherapy. Neurosurgery was offered to selected patients. Radiotherapy generally was used only as a salvage therapy after chemotherapy failure. The primary outcome measure was overall survival and was depicted using a Kaplan-Meier plot. RESULTS: The 3-year overall survival rates were 38% for the whole cohort, 69% for those who presented with brain metastases at diagnosis (group 1), and 21% and 0% for those who developed metastases after initial chemotherapy (group 2) and while receiving chemotherapy (group 3), respectively. For the whole cohort, the median overall survival was 10.6 months (range, 5.5 months to not evaluable); and, for groups 1, 2, and 3 individually, the overall survival was not yet reached (range, from 7.4 months to not evaluable), 6.2 months (range, 2.1-15.3 months), and 2.7 months (range, from 0.6 months to not evaluable), respectively. The 3-year survival rate for those who received GAMEC chemotherapy was 56% compared with 27% for those who received chemotherapy pre-GAMEC. CONCLUSIONS: The prognosis for patients with germ cell tumors and brain metastases seems less bleak than previously thought. It is possible to achieve long-term survival with chemotherapy alone.

Patient-derived xenografts for individualized care in advanced sarcoma.

BACKGROUND: Patients with advanced, metastatic sarcoma have a poor prognosis, and the overall benefit from the few standard-of-care therapeutics available is small. The rarity of this tumor, combined with the wide range of subtypes, leads to difficulties in conducting clinical trials. The authors previously reported the outcome of patients with a variety of common solid tumors who received treatment with drug regimens that were first tested in patient-derived xenografts using a proprietary method ("TumorGrafts"). METHODS: Tumors resected from 29 patients with sarcoma were implanted into immunodeficient mice to identify drug targets and drugs for clinical use. The results of drug sensitivity testing in the TumorGrafts were used to personalize cancer treatment. RESULTS: Of 29 implanted tumors, 22 (76%) successfully engrafted, permitting the identification of treatment regimens for these patients. Although 6 patients died before the completion of TumorGraft testing, a correlation between TumorGraft results and clinical outcome was observed in 13 of 16 (81%) of the remaining individuals. No patients progressed during the TumorGraft-predicted therapy. CONCLUSIONS: The current data support the use of the personalized TumorGraft model as an investigational platform for therapeutic decision-making that can guide treatment for rare tumors such as sarcomas. A randomized phase 3 trial versus physician's choice is warranted.

The prognostic role of circulating tumor cells in heavily pretreated individuals with a low life expectancy.

AIMS: Studies of circulating tumor cells (CTCs) have generally recruited individuals with newly diagnosed metastatic cancer, with recent data also indicating their prognostic value in the adjuvant setting. Their role in dying patients has not been established. EXPERIMENTAL: CTCs were measured in 43 individuals with metastatic breast cancer estimated to have less than 6 months to live who had exhausted standard therapeutic options. RESULTS: Those with a CTC count of ≤ 100 had a median of 182 days to live, compared with those with a CTC count of >100 who had a median of 17 days until death (p = 0.009, log rank, HR: 3.1, 95% CI: 1.4-7.3). CONCLUSION: A CTC count of >100 is associated with imminent death. Provided external validity is demonstrated, such information would be useful for patients and their families who often request specific prognostic clarity and could improve the quality of end-of-life care.

Novel cancer therapies: treatments driven by tumour biology.

The traditional view is that treatments within oncology largely consist of chemotherapy, which aims to maximise damage to the rapidly dividing cancer cells but often at the expense of normal cells and overall quality of life for the patient. The development of anticancer drugs has changed from the serendipitous discoveries of the past, to today's purposeful targeting of cancer cells which takes advantage of novel technological developments and a greater understanding of tumour biology. The aim of these new treatments is to affect the essential function of the cancer cell while sparing normal cells, and limiting side effects. The phenotypic characteristics of tumours, such as unregulated growth signalling, development of new vascular systems and the evasion of immune destruction are used to identify potential drug targets. Here we review the clinical evidence and molecular mechanisms for novel therapies that are currently in use and those that are in development.

Embolization of uterine arteriovenous malformations in patients with gestational trophoblastic tumors: a review of patients at Charing Cross Hospital, 2000-2009.

OBJECTIVE: To review the indications, efficacy and follow-up for gestational trophoblastic tumor (GTT) patients treated for uterine arteriovenous vascular malformations (AVMs) and bleeding vaginal metastases with modern polyvinyl alcohol particle (PVA)-based radiological embolization. STUDY DESIGN: GTT patients undergoing embolization were identified from the Charing Cross Hospital database. The patients' records were assessed for indication, technique used, primary and overall success in controlling bleeding, complications and subsequent pregnancy outcome. RESULTS: During the period 2000-2009, 19 patients were treated for persistent or life-threatening bleeding by PVA-based uterine artery embolization performed via the femoral artery approach. Embolization resulted in control of hemorrhage in 18 of the 19 patients; 15 achieved control after the first procedure, with only 4 patients requiring a second procedure. In 1 case surgical intervention was required to control bleeding. The most frequent morbidity from the procedure was pelvic pain, requiring opiate administration; there were no other regular complications. The fertility outcome for these 19 patients indicates that 9 women have gone on to deliver a total of 12 healthy infants postembolization. CONCLUSION: For GTT patients with heavy bleeding from AVMs, uterine artery embolization is a safe and effective treatment with low short-term toxicity and no obvious detrimental effect on future fertility.

Signal peptidase I-mediated processing of an engineered mammalian cytochrome b(5) precursor is an exocytoplasmic post-translocational event in Escherichia coli.

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane. The temporal nature of cleavage of the signal sequence during pre-protein translocation has remained elusive. Using an engineered mammalian cytochrome b(5) precursor we demonstrate that the signal peptide processing in Escherichia coli is an event that can occur after almost complete exocytoplasmic translocation of the preprotein is accomplished. We discuss implications of the findings in light of the known working model of sec-dependent pre-protein translocon.

Harms of cigarette smoking: call to increase knowledge among children.

Although the major burden of disease caused by smoking is observed in the adult population, two-third of smokers start before the age of 18 years. Reducing the number of young smokers could lead to marked improvements to the health of the UK population and save billions of pounds in National Health Service finances. However, very little is known about what makes children decide to not smoke or to quit early. We believe that increased awareness of the health risks associated with smoking will reduce smoking uptake among children. This study identifies a significant lack of knowledge among children aged 11-17 years at two London secondary schools and potentially identifies an area for improving our antismoking programmes. Although 80% of pupils cited lung cancer as being a smoking-related disease, very few other conditions could be recalled. We must do all we can to reduce smoking uptake in children. Understanding their baseline knowledge is the first step towards addressing the deficits in our current antismoking programmes.

A comparison of methods for shoulder strength assessment and analysis of Constant score change in patients aged over fifty years in the United Kingdom.

Normal values for shoulder assessment are imperative for the diagnosis of pathology and measurement of treatment outcome. Normal values for the United Kingdom are currently not known. This study comprised 108 control patients aged over 50 years who were under the care of one general practitioner, and the Constant score was measured. The Constant score was assessed via 3 techniques for strength measurement: maximum strength with myometer, mean strength with myometer, and maximum strength with fixed spring balance. Myometer maximum strength values were very similar to fixed spring balance maximum strength values, with a mean difference of 0.5. Myometer mean strength was lower than myometer maximum strength by 3 points. Age and sex both significantly affected Constant score (P < .001 for both). The Constant score fell by 0.3 points per year over 50 years of age. Men had a score that was 7.5 points greater than that in women. The Constant score decreases predictably with age in the United Kingdom. Methods of strength assessment are not the same. A uniform method of shoulder strength assessment is required to allow for meaningful comparisons between series.

The need for multidisciplinarity in specialist training to optimize future patient care.

Harmonious interactions between radiation, medical, interventional and surgical oncologists, as well as other members of multidisciplinary teams, are essential for the optimization of patient care in oncology. This multidisciplinary approach is particularly important in the current landscape, in which standard-of-care approaches to cancer treatment are evolving towards highly targeted treatments, precise image guidance and personalized cancer therapy. Herein, we highlight the importance of multidisciplinarity and interdisciplinarity at all levels of clinical oncology training. Potential deficits in the current career development pathways and suggested strategies to broaden clinical training and research are presented, with specific emphasis on the merits of trainee involvement in functional multidisciplinary teams. Finally, the importance of training in multidisciplinary research is discussed, with the expectation that this awareness will yield the most fertile ground for future discoveries. Our key message is for cancer professionals to fulfil their duty in ensuring that trainees appreciate the importance of multidisciplinary research and practice.

LMTK3 escapes tumour suppressor miRNAs via sequestration of DDX5.

Lemur tyrosine kinase-3 (LMTK3) plays an important role in cancer progression and is associated with breast, lung, gastric and colorectal cancer. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that typically repress target genes at post-transcriptional level and have an important role in tumorigenesis. By performing a miRNA expression profile, we identified a subset of miRNAs modulated by LMTK3. We show that LMTK3 induces miR-34a, miR-196-a2 and miR-182 levels by interacting with DEAD-box RNA helicase p68 (DDX5). LMTK3 binds via DDX5 to the pri-miRNA of these three mature miRNAs, thereby sequestrating them from further processing. Ectopic expression of miR-34a and miR-182 in LMTK3-overexpressing cell lines (MCF7-LMTK3 and MDA-MB-231-LMTK3) inhibits breast cancer proliferation, invasion and migration. Interestingly, miR-34a and miR-182 directly bind to the 3'UTR of LMTK3 mRNA and consequently inhibit both its stability and translation, acting as tumour suppressor-like miRNAs. In aggregate, we show that LMTK3 is involved in miRNA biogenesis through modulation of the Microprocessor complex, inducing miRNAs that target LMTK3 itself.

Noncoding RNAs and the control of hormonal signaling via nuclear receptor regulation.

Despite its identification over 100 years ago, new discoveries continue to add to the complexity of the regulation of the endocrine system. Today the nuclear receptors (NRs) that play such a pivotal role in the extensive communication networks of hormones and gene expression remain an area of intense research. By orchestrating core processes, from metabolism to organismal development, the gene expression programs they control are dependent on their cellular context, their own levels, and those of numerous co-regulatory proteins. A previously unknown component of these networks, noncoding RNAs (ncRNAs) are now recognized as potent regulators of NR signaling, influencing receptor and co-factor levels and functions while being reciprocally regulated by the NRs themselves. This review explores the regulation enacted by microRNAs and long ncRNAs on NR function, using representative examples to show the varied roles of ncRNAs, in turn producing significant effects on the NR functional network in health and disease.

Growth arrest-specific transcript 5 associated snoRNA levels are related to p53 expression and DNA damage in colorectal cancer.

BACKGROUND: The growth arrest-specific transcript 5 gene (GAS5) encodes a long noncoding RNA (lncRNA) and hosts a number of small nucleolar RNAs (snoRNAs) that have recently been implicated in multiple cellular processes and cancer. Here, we investigate the relationship between DNA damage, p53, and the GAS5 snoRNAs to gain further insight into the potential role of this locus in cell survival and oncogenesis both in vivo and in vitro. METHODS: We used quantitative techniques to analyse the effect of DNA damage on GAS5 snoRNA expression and to assess the relationship between p53 and the GAS5 snoRNAs in cancer cell lines and in normal, pre-malignant, and malignant human colorectal tissue and used biological techniques to suggest potential roles for these snoRNAs in the DNA damage response. RESULTS: GAS5-derived snoRNA expression was induced by DNA damage in a p53-dependent manner in colorectal cancer cell lines and their levels were not affected by DICER. Furthermore, p53 levels strongly correlated with GAS5-derived snoRNA expression in colorectal tissue. CONCLUSIONS: In aggregate, these data suggest that the GAS5-derived snoRNAs are under control of p53 and that they have an important role in mediating the p53 response to DNA damage, which may not relate to their function in the ribosome. We suggest that these snoRNAs are not processed by DICER to form smaller snoRNA-derived RNAs with microRNA (miRNA)-like functions, but their precise role requires further evaluation. Furthermore, since GAS5 host snoRNAs are often used as endogenous controls in qPCR quantifications we show that their use as housekeeping genes in DNA damage experiments can lead to inaccurate results.

Emerging roles of competing endogenous RNAs in cancer: insights from the regulation of PTEN.

The capacity of noncoding RNA to regulate gene expression in health and disease is epitomized by the microRNAs, small ∼22-nucleotide RNAs that target mRNAs to repress their translation into protein. Recently a previously unrecognized gene regulatory layer has emerged, characterized by the ability of a wide range of RNA transcripts to vie for microRNA binding and alleviate the repressive effect of microRNAs on their mRNA targets. Termed competing endogenous RNAs (ceRNAs), these participate in a microRNA-dependent cross talk, producing robust networks that when perturbed may lead to cancer. To date, the tumor suppressor PTEN has been most extensively validated as competing with a variety of ceRNAs in different cancers: reducing these ceRNAs appears to reduce PTEN levels, tipping cells toward cancer progression. In this review we look at ceRNA networks in cancer, their characteristics, and constituent parts, focusing on the insights that can be gained from the studies conducted on PTEN. We also explore the conditions that facilitate ceRNA cross talk, proposing that the disruption of these conditions may represent a general phenomenon in carcinogenesis.

The p53 miRNA interactome and its potential role in the cancer clinic.

p53 is one of the most frequently mutated tumor suppressors. It regulates protein-coding genes and noncoding RNAs involved in many cellular processes, functioning predominantly at the transcriptional level but also through nontranscriptional processes. miRNAs have recently been identified as key mediators of the p53 stress-response pathway. p53 regulates miRNA transcription and processing, and miRNAs regulate p53 activity and expression and, accordingly, various feedback/feed-forward loops have been identified. Many chemotherapeutic agents induce cancer cell death or senescence via DNA damage and the subsequent activation of p53. Resistance to chemotherapy can occur due to the mutation of components in p53 signaling networks. A better understanding of the role of the various components within these pathways and their interactions with each other may allow the modification and improvement of current treatments, and the design of novel therapies. Improving our knowledge of the role of miRNAs in such p53 signaling networks may be crucial to achieving this.