Role of the unique N-terminal domain of CtBP2 in determining the subcellular localisation of CtBP family proteins

BACKGROUND: CtBP1 and CtBP2 are transcriptional co-repressors that modulate the activity of a large number of transcriptional repressors via the recruitment of chromatin modifiers. Many CtBP-regulated proteins are involved in pathways associated with tumorigenesis, including TGF-beta and Wnt signalling pathways and cell cycle regulators such as RB/p130 and HDM2, as well as adenovirus E1A. CtBP1 and CtBP2 are highly similar proteins, although evidence is emerging that their activity can be differentially regulated, particularly through the control of their subcellular localisation. CtBP2s from diverse species contain a unique N-terminus, absent in CtBP1 that plays a key role in controlling the nuclear-cytoplasmic distribution of the protein.RESULTS: Here we show that amino acids (a.a.) 4-14 of CtBP2 direct CtBP2 into an almost exclusively nuclear distribution in cell lines of diverse origins. Whilst this sequence contains similarity to known nuclear localisation motifs, it cannot drive nuclear localisation of a heterologous protein, but rather has been shown to function as a p300 acetyltransferase-dependent nuclear retention sequence. Here we define the region of CtBP2 required to co-operate with a.a. 4-14 to promote CtBP2 nuclear accumulation as being within a.a. 1-119. In addition, we show that a.a. 120-445 of CtBP2 can also promote CtBP2 nuclear accumulation, independently of a.a. 4-14. Finally, CtBP1 and CtBP2 can form heterodimers, and we show that the interaction with CtBP2 is one mechanism whereby CtBP1 can be recruited to the nucleus.CONCLUSION: Together, these findings represent key distinctions in the regulation of the functions of CtBP family members that may have important implications as to their roles in development, and cell differentiation and survival.<br/

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head &amp; neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p &lt; 0.05). We used TGF-? treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ? 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure

Investigation of a novel protein-protein interaction involving the hdm2 oncoprotein

The p53 tumor suppressor protein has a critical role in the cellular response to stress, and when activated, functions to transactivate genes which induce apoptosis or growth arrest.  The hdm2 oncoprotein is the most significant regulator of p53 activity, and it achieves this partly through inhibition of p53 transactivating ability.  Hdm2 also interacts with other non-p53 proteins and through its many protein-protein interactions, influences diverse cellular pathways.  Work by our group previously identified a putative protein-protein interaction between hdm2 and the redox-sensitive transcriptional co-repressor CtBP2.  Based on this observation, the objectives of the current study were:  (i) To verify that hdm2 and CtBP2 interact, (ii) To characterise the physical nature of the interaction, (iii) to determine the functional consequences of the interaction and how it may be regulated. The interaction between hdm2 and CtBP2 was verified using in vitro and in vivo protein binding assays.  Deletion mutants were used to identify the interacting domains in the proteins, and the effect of the interaction on p53 activity assessed using reporter gene assays. Hdm2 participates in a novel protein-protein interaction with CtBP2 both in vitro and in vivo.  The acidic domain of hdm2 and the N-terminus of CtBP2 are necessary and sufficient for this interaction.  CtBP proteins undergo an NADH-induced conformational change, which we show results in a loss of its hdm2 binding ability.  This negative regulation is dependent on the conserved NADH-binding GXGXXG motif in CtBP2. The recruitment of CtBP2 by hdm2 results in a promoter selective repression of p53-depdnent transcription. Furthermore, hypoxia-mimicking conditions which increase intracellular NADH levels abolish this repression, thus enhancing p53 activity.</p

Investigation of a novel protein : protein interaction involving the hdm2 oncoprotein

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Increased local recurrence and reduced survival from colorectal cancer following anastomotic leak: systematic review and meta-analysis

Objective: To examine the long-term oncological impact of anastomotic leakage (AL) after restorative surgery for colorectal cancer using meta-analytical methods. Outcomes evaluated were local recurrence, distant recurrence, and survival.Background: Recurrence after potentially curative surgery for colorectal cancer remains a significant clinical problem and has a poor prognosis. AL may be a risk factor for disease recurrence, however available studies have been conflicting. A meta-analysis was conducted to investigate the impact of AL on disease recurrence and long-term survival.Methods: Studies published between 1965 and 2009 evaluating the long-term oncological impact of AL were identified by an electronic literature search. Outcomes evaluated included local recurrence, distant recurrence, and cancer specific survival. Meta-analysis was performed using the DerSimonian-Laird random-effects model to compute odds ratio and 95% confidence intervals. Study heterogeneity was evaluated using Q statistics and I2 and publication bias assessed with funnel plots and Egger's test.Results: Twenty-one studies comprising 13 prospective nonrandomized studies, 1 prospective randomized, and 7 retrospective studies met the inclusion criteria, yielding a total of 21,902 patients. For rectal anastomoses, the odd ratios (OR) of developing a local recurrence when there was AL was 2.05 (95% CI = 1.51-2.8; P = 0.0001). For studies describing both colon and rectal anastomoses, the OR of local recurrence when there was an AL was 2.9 (95% CI = 1.78-4.71; P &lt; 0.001). The OR of developing a distant recurrence after AL was 1.38 (95% CI = 0.96-1.99; P = 0.083). Long term cancer specific mortality was significantly higher after AL with an OR of 1.75 (95% CI = 1.47-2.1; P = 0.0001).Conclusions: AL has a negative prognostic impact on local recurrence after restorative resection of rectal cancer. A significant association between colorectal AL and reduced long-term cancer specific survival was also noted. No association between AL and distant recurrence was found.<br/

Network mapping of molecular biomarkers influencing radiation response in rectal cancer

Preoperative radiotherapy (RT) plays an important role in the management of locally advanced rectal cancer (RC). Tumor regression after RT shows marked variability, and robust molecular methods are needed to help predict likely response. The aim of this study was to review the current published literature and use Gene Ontology (GO) analysis to define key molecular biomarkers governing radiation response in RC. A systematic review of electronic bibliographic databases (Medline, Embase) was performed for original articles published between 2000 and 2015. Biomarkers were then classified according to biological function and incorporated into a hierarchical GO tree. Both significant and nonsignificant results were included in the analysis. Significance was binarized on the basis of univariate and multivariate statistics. Significance scores were calculated for each biological domain (or node), and a direct acyclic graph was generated for intuitive mapping of biological pathways and markers involved in RC radiation response. Seventy-two individual biomarkers across 74 studies were identified. On highest-order classification, molecular biomarkers falling within the domains of response to stress, cellular metabolism, and pathways inhibiting apoptosis were found to be the most influential in predicting radiosensitivity. Homogenizing biomarker data from original articles using controlled GO terminology demonstrated that cellular mechanisms of response to RT in RC—in particular the metabolic response to RT—may hold promise in developing radiotherapeutic biomarkers to help predict, and in the future modulate, radiation response.</p

Hdm2 recruits a hypoxia-sensitive corepressor to negatively regulate p53-dependent transcription

The transcription factor p53 lies at the center of a protein network that controls cell cycle progression and commitment to apoptosis. p53 is inactive in proliferating cells, largely because of negative regulation by the Hdm2/Mdm2 oncoprotein, with which it physically associates. Release from this negative regulation is sufficient to activate p53 and can be triggered in cells by multiple stimuli through diverse pathways. This diversity is achieved in part because Hdm2 uses multiple mechanisms to inactivate p53; it targets p53 for ubiquitination and degradation by the proteosome, shuttles it out of the nucleus and into the cytoplasm, prevents its interaction with transcriptional coactivators, and contains an intrinsic transcriptional repressor activity. Here we show that Hdm2 can also repress p53 activity through the recruitment of a known transcriptional corepressor, hCtBP2. This interaction, and consequent repression of p53-dependent transcription, is relieved under hypoxia or hypoxia-mimicking conditions that are known to increase levels of intracellular NADH. CtBP proteins can undergo an NADH-induced conformational change, which we show here results in a loss of their Hdm2 binding ability. This pathway represents a novel mechanism whereby p53 activity can be induced by cellular stress.<br/

Metabolic syndrome is a predictor of all site and liver-specific recurrence following primary resection of colorectal cancer: Prospective cohort study of 1006 patients

Introduction: Large epidemiological studies have demonstrated the link between metabolic syndrome and cancer development, including colorectal cancer. However, the influence of metabolic syndrome on disease progression is less well studied, particularly in the post-surgical setting. This study investigates the effect of metabolic syndrome on colorectal cancer recurrence (all-site and liver-specific) after curative surgery for Stage I-III disease. Materials and methods: Consecutive patients who underwent curative resection for Stage I-III colorectal cancer in a single UK centre were prospectively recruited. Disease-free and overall survival with metabolic syndrome as a factor, were determined using the Kaplan-Meier technique. Hazard ratios for all-site and liver-specific recurrence were determined using univariable and multivariable Cox-regression models. Results: 1006 patients were recruited and followed up for a median of 50 months (IQR 30–67). 177 patients (17.6%) met the criteria for metabolic syndrome. 245 patients (25.4%) developed recurrence, 161 (16.0%) of these had liver recurrence. The presence of metabolic syndrome was associated with a reduction in disease-free survival from 69 to 58 months (p &lt; 0.001) and overall survival from 74 to 61 months (p &lt; 0.001). Metabolic syndrome was an independent predictor of all-site (HR 1.76; p &lt; 0.001) and liver-specific (HR 1.74; p = 0.01) recurrence. Conclusion: Metabolic syndrome is a predictor of all-site and liver-specific recurrence after primary resection of stage I-III colorectal cancer.</p

Pelvic exenteration with en bloc resection of the pelvic sidewall and intraoperative electron beam radiotherapy with Mobetron® for locally advanced rectal cancer

Locally advanced rectal cancer (LARC) is defined as a tumour which is predicted by magnetic resonance imaging to require an extended surgical resection beyond the total mesorectal excision plane. Preoperative neoadjuvant treatments are commonly utilised to downstage and downsize the tumour, facilitating resection. In patients with persistent predicted involved margins or poor response to neoadjuvant treatment, several studies and a meta-analysis have shown that intraoperative electron beam radiotherapy (IOERT) is a further useful adjunct to extended margin surgery, leading to low recurrence within the IOERT field even in patients with a positive margin. Here we describe a case of LARC with anterior and pelvic sidewall involvement and predicted stage of T4N0M0 with involved circumferential resection margins. A poor radiological response to neoadjuvant therapy was noted, and the patient was subsequently treated with a posterior pelvic exenteration with en bloc pelvic sidewall resection and IOERT using the IntraOp®Mobetron® device (IntraOp, Sunnyvale, CA, USA). Final histology was ypT4N0 EMVI-negative R1 (pelvic sidewall margin, 0.3 mm) TRG4 (minimal response to neoadjuvant therapy). Post-operative recovery was complicated by a temporary ileus requiring parenteral nutrition. At 6-week and 3-month reviews, the patient had returned to preoperative functional status

Stem cell-like breast cancer cells with acquired resistance to metformin are sensitive to inhibitors of NADH-dependent CtBP dimerisation

Altered flux through major metabolic pathways is a hallmark of cancer cells, and provides opportunities for therapy. Stem cell-like cancer (SCLC) cells can cause metastasis and therapy resistance. They possess metabolic plasticity, theoretically enabling resistance to therapies targeting a specific metabolic state. The CtBP transcriptional regulators are potential therapeutic targets in highly glycolytic cancer cells, as they are activated by the glycolytic coenzyme NADH. However, SCLC cells commonly exist in an oxidative state with low rates of glycolysis. Metformin inhibits complex I of the mitochondrial electron transport chain; it can kill oxidative SCLC cells, and has anti-cancer activity in patients. SCLC cells can acquire resistance to metformin through increased glycolysis. Given the potential for long-term metformin therapy, we have studied acquired metformin resistance in cells from the claudin-low subtype of breast cancer. Cells cultured for 8 weeks in sub-IC-50 metformin concentration proliferated comparably to untreated cells, and exhibited higher rates of glucose uptake. SCLC cells were enriched in metformin-adapted cultures. These SCLC cells acquired sensitivity to multiple methods of inhibition of CtBP function, including a cyclic peptide inhibitor of NADH-induced CtBP dimerisation. Single cell mRNA sequencing identified a reprogramming of epithelial mesenchymal and stem cell gene expression in the metformin-adapted SCLC cells. These SCLC cells demonstrated an acquired dependency on one of these genes, Tenascin C. Thus, in addition to acquisition of sensitivity to glycolysis-targeting therapeutic strategies, the reprograming of gene expression in the metformin-adapted SCLC cells renders them sensitive to potential therapeutic approaches not directly linked to cell metabolism