Metabolomic Fingerprinting for the Detection of Early-Stage Lung Cancer: From the Genome to the Metabolome.

The five-year survival rate of lung cancer patients is very low, mainly because most newly diagnosed patients present with locally advanced or metastatic disease. Therefore, early diagnosis is key to the successful treatment and management of lung cancer. Unfortunately, early detection methods of lung cancer are not ideal. In this brief review, we described early detection methods such as chest X-rays followed by bronchoscopy, sputum analysis followed by cytological analysis, and low-dose computed tomography (LDCT). In addition, we discussed the potential of metabolomic fingerprinting, compared to that of other biomarkers, including molecular targets, as a low-cost, high-throughput blood-based test that is both feasible and affordable for early-stage lung cancer screening of at-risk populations. Accordingly, we proposed a paradigm shift to metabolomics as an alternative to molecular and proteomic-based markers in lung cancer screening, which will enable blood-based routine testing and be accessible to those patients at the highest risk for lung cancer.

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

Prognostic Value of Molecular Detection of Lymph Node Metastases After Curative Resection of Stage II Colon Cancer: A Systematic Pooled Data Analysis.

BACKGROUND: We aimed to clarify the prognostic value of guanylyl cyclase C (GCC) lymph node ratio (LNR) status as a predictor of recurrence in untreated stage IIA colon cancer on the basis of pooled individual data from previous studies. METHODS: Patients were classified according to predefined GCC LNR risk groups (low, LNR ≤ 0.1; intermediate, 0.1 < LNR ≤ 0.2; high, LNR > 0.2). Outcomes included time to recurrence, disease-free survival, and overall survival. Stratified log-rank tests and multivariate Cox models assessed the association between outcomes and GCC lymph node status. RESULTS: The final data set contained 553 patients with stage IIA colon cancer with a median of 18 lymph nodes examined after resection; 65 patients (11.8%) had recurrence. Overall, 109 patients (19.7%) were classified high risk on the basis of GCC LNR. In multivariate analysis, high GCC LNR value (> 0.2) was a significant predictor of cancer recurrence (hazard ratio [HR], 3.18; 95% confidence interval [CI], 1.77-5.71; P < .001) and lower disease-free survival (HR, 2.40; 95% CI, 1.60-3.62; P < .001) and overall survival (HR, 2.12; 95% CI, 1.35-3.33; P = .001). CONCLUSION: Patients considered at high risk on the basis of their GCC LNR status have significantly inferior outcomes compared to those with low GCC LNR values, particularly among those traditionally considered to be at low risk for recurrence.

Molecular testing for lymph node metastases as a determinant of colon cancer recurrence: results from a retrospective multicenter study.

PURPOSE: Recurrence risk assessment to make treatment decisions for early-stage colon cancer patients is a major unmet medical need. The aim of this retrospective multicenter study was to evaluate the clinical utility of guanylyl cyclase C (GCC) mRNA levels in lymph nodes on colon cancer recurrence. METHODS: The proportion of lymph nodes with GCC-positive mRNA (LNR) was evaluated in 463 untreated T3N0 patients, blinded to clinical outcomes. One site's (n = 97) tissue grossing method precluded appropriate lymph node assessment resulting in post hoc exclusion. Cox regression models tested the relationship between GCC and the primary endpoint of time to recurrence. Assay methods, primary analyses, and cut points were all prespecified. RESULTS: Final dataset contained 366 patients, 38 (10%) of whom had recurrence. Presence of four or more GCC-positive lymph nodes was significantly associated with risk of recurrence [hazard ratio (HR) = 2.46, 95% confidence interval (CI), 1.07-5.69, P = 0.035], whereas binary GCC LNR risk class (HR = 1.87, 95% CI, 0.99-3.54, P = 0.054) and mismatch repair (MMR) status (HR = 0.77, 95% CI, 0.36-1.62, P = 0.49) were not. In a secondary analysis using a 3-level GCC LNR risk group classification of high (LNR > 0.20), intermediate (0.10 < LNR ≤ 0.20), and low (LNR ≤ 0.10), high-risk patients had a 2.5 times higher recurrence risk compared with low-risk patients (HR = 2.53, 95% CI, 1.24-5.17, P = 0.011). CONCLUSIONS: GCC status is a promising prognostic factor independent of traditional histopathology risk factors in a contemporary population of patients with stage IIa colon cancer not treated with adjuvant therapy, but GCC determination must be performed with methodology adapted to the tissue procurement and fixation technique.

Affinity-based assays for the identification and quantitative evaluation of noncovalent poly(ADP-ribose)-binding proteins.

Poly(ADP-ribose) polymerases have been linked to several cellular functions, most of which being mediated through the dynamics of poly(ADP-ribose) (pADPr). In several pathways, pADPr is the effector molecule that regulates cellular signaling and dictates biological outcomes. pAPDr is a central molecule that is capable of promoting both cell survival through the maintenance of genome integrity and cell death that occurs by way of a signal-mediated apoptotic-like process. Thus, interactions with pADPr are extremely important in bringing about the balanced regulation that controls cell fate. Further clues regarding these functions are emerging from a growing list of proteins with which pADPr interacts. Here, we describe the current approaches for investigating noncovalent protein interactions with pADPr.

Iduna protects the brain from glutamate excitotoxicity and stroke by interfering with poly(ADP-ribose) polymer-induced cell death.

Glutamate acting on N-methyl-D-aspartate (NMDA) receptors induces neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 (PARP-1) and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a previously undescribed NMDA receptor-induced survival protein that is neuroprotective against glutamate NMDA receptor-mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer-induced cell death (parthanatos). Iduna's protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer-binding protein, and mutation at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion-induced stroke in mice. To our knowledge, these results define Iduna as the first known endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling could be a new therapeutic strategy for the treatment of neurologic disorders.

Evaluation of guanylyl cyclase C lymph node status for colon cancer staging and prognosis.

PURPOSE: The prognostic significance of guanylyl cyclase C (GCC) gene expression in lymph nodes (LNs) was evaluated in patients with stage II colon cancer who were not treated with adjuvant chemotherapy. We report a planned analysis performed on 241 patients. METHODS: GCC mRNA was quantified by RT-qPCR using formalin-fixed LN tissues from patients with untreated stage II colon cancer who were diagnosed from 1999-2006 with at least ten LNs examined and blinded to clinical outcomes. Lymph node ratio (LNR) is the number of GCC-positive nodes divided by total number of informative LNs. Risk categories of low (0-0.1) and high (>0.1) for LNR were chosen by significance using Cox regression models. The data were tested for association with time to recurrence. RESULTS: Twenty-nine patients (12%) had a disease recurrence or cancer death. The LNR significantly predicted higher recurrence risk for 84 patients (34.9%) classified as high risk (hazard ratio (HR), 2.38; P=0.02). The estimated 5-year recurrence rates were 10% and 27% for the low- and high-risk groups, respectively. After adjusting for age, T stage, number of nodes assessed, and MMR status, a significant association remained (HR, 2.61; P=0.02). In a subset of patients (n=181) with T3 tumor, ≥12 nodes examined and negative margins, a significant association between the GCC LNR and recurrence risk also was observed (HR, 5.06; P=0.003). CONCLUSIONS: Our preliminary results suggest that detection of GCC mRNA in LNs is associated with risk of disease recurrence in patients with untreated stage II colon cancer. A larger validation study is ongoing.

Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos).

The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. We show that AIF is a high-affinity poly(ADP-ribose) (PAR)-binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF's DNA binding site, and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH (reduced form of nicotinamide adenine dinucleotide) oxidase activity, its ability to bind FAD (flavin adenine dinucleotide) or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death after PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF's bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide notable opportunities for preventing cell death after activation of PARP-1.

Comparison of histopathology and RT-qPCR amplification of guanylyl cyclase C for detection of colon cancer metastases in lymph nodes.

AIMS: In colorectal cancer (CRC), the presence of lymph node (LN) metastases is an important prognostic factor. Approximately 20% of patients diagnosed as having node-negative (pN0) CRC will relapse. Pathological nodal stage misclassification due to sampling error resulting from the small volume of tissue tested has been proposed to explain this recurrence rate in pN0 patients. The authors compared the assessment of node positivity by histopathology (HP) with a molecular method which can accommodate larger tissue volumes. METHODS: Detection rate of guanylyl cyclase C (GCC) mRNA was determined in 1,495 LNs from 99 CRC patients. Using a subset of 647 LNs, multiple levels of HP analysis were compared with GCC mRNA molecular detection. Finally, clinicopathological factors were correlated with the molecular detection of GCC and clinical outcome in 123 patients with pN0 colon cancer. RESULTS: GCC mRNA was detected in 8.0% of the 560 nodes initially identified as HP-negative, whereas two repeat HP examinations detected 3.0% of these cases. In HP-positive LNs, the GCC mRNA detection rate was 90% (78/87) when half-LN were tested. Testing the entire LN remaining after HP by GCC increased the detection rate of HP-positive LNs to 95% (p=0.027). In comparison, 75% (65/87) and 92% (80/87) of the LN positive by clinical HP remained positive when one or two subsequent sections were examined by HP. Finally, patients with pN0 disease who were GCC-positive exhibited an earlier time of recurrence (hazard ratio, 3.54; 95% CI 1.40 to 8.98; p=0.0077). CONCLUSIONS: Molecular detection of tumour cells in LNs may have prognostic value in identifying patients diagnosed as having pN0 colon cancer who will relapse following surgery.

Analytical performance of a qRT-PCR assay to detect guanylyl cyclase C in FFPE lymph nodes of patients with colon cancer.

Up to 30% of patients with stage II (pN0) colon cancer develop recurrences, suggesting that the presence of lymph node (LN) metastases escaped detection at histopathologic staging. A simple way to overcome this limitation and to improve staging accuracy is to use reverse transcription-polymerase chain reaction (RT-PCR) to examine a larger fraction or an entire specimen. The Guanylyl cyclase C (GCC) gene is uniquely expressed in apical cells of the gastrointestinal tract. Its expression in colon cancer cells and metastases is conserved. Therefore, detection of GCC mRNA in LNs has been shown to be indicative of the presence of colon cancer metastases. As the current processing of LNs involves formalin fixation and paraffin embedding, we developed a method for extracting RNA from formalin-fixed paraffin-embedded LN specimens and detecting GCC mRNA by quantitative RT-PCR. The assay has a dynamic range of 5 logs, an average amplification efficiency of 98.4% (95% confidence interval, 96.6-100.3), a reaction linearity of 0.998 (95% confidence interval, 0.997-0.999), and also intraplate and interplate CVs of <1% and <5%, respectively. The test specificity was 98% with LNs collected from patients affected by conditions other than colon cancer (n=380). Sensitivity was 97% for patients with stage III colon cancer (n=34), whereas 35% of patients with stages I and II disease (n=51) had at least 1 GCC mRNA-positive LN. The high specificity of GCC mRNA suggests that routine utilization of the quantitative RT-PCR test has the potential to improve the detection of colon cancer metastases in LNs.

PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Ataxia telangiectasia mutated (ATM) signaling network is modulated by a novel poly(ADP-ribose)-dependent pathway in the early response to DNA-damaging agents.

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling.

Dynamic relocation of poly(ADP-ribose) glycohydrolase isoforms during radiation-induced DNA damage.

Poly(ADP-ribosyl)ation is a very early cellular response to DNA damage. Poly(ADP-ribose) (PAR) accumulation is transient since PAR is rapidly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). PARG may play a prominent role in DNA damage response and repair by removing PAR from modified proteins including PARP-1. Using living cells, we provide evidence that in response to DNA damage induced by gamma-irradiation the cytoplasmic 103 kDa PARG isoform translocates into the nucleus. We further observed that the nuclear GFP-hPARG110 enzyme relocalizes to the cytoplasm in response to DNA damage. Using different GFP-PARG fusion proteins specific for the nuclear and cytoplasmic forms, we demonstrate their dynamic distribution between cytoplasm and nucleoplasm and a high mobility of major PARG isoforms by fluorescence recovery after photobleaching (FRAP). The dynamic relocation of all PARG isoforms presented in this report reveals a novel biological mechanism by which PARG could be involved in DNA damage response.

Targeting poly(ADP-ribosyl)ation: a promising approach in cancer therapy.

Recent progress in the field of DNA repair has demonstrated that transient inhibition of DNA damage detection or repair using potent poly(ADP-ribose) polymerase (PARP) inhibitors could improve the efficacy of cancer treatments. Although more study is needed, recent publications lead to optimism that the inhibition of poly(ADP-ribose) synthesis could selectively kill cancer cells when used to treat tumours with defective BRCA proteins. These reports and others shed some light on the DNA damage signalling and repair processes involving PARPs. However, a better understanding of the molecular mechanisms regulated by poly(ADP-ribose) metabolism will be essential before optimism can be replaced by clinical realization.

Nonisotopic methods for determination of poly(ADP-ribose) levels and detection of poly(ADP-ribose) polymerase.

Poly(ADP-ribosyl)ation is a post-translational modification catalyzed mostly by the 116-kDa enzyme poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that transfers an ADP-ribose moiety onto a limited number of nuclear proteins, including itself. When cells are exposed to environmental stresses such as alkylating agents or free radicals, there is up to a 500-fold increase in net poly(ADP-ribose) synthesis in response to DNA strand breaks. The enzyme responsible for 80% to 90% of this stimulated poly(ADP-ribose) synthesis is PARP-1, while other PARPs are responsible for the remaining 10% to 20%. The physiological meaning of these phenomena is not clear; however, it can be interpreted as a way of translating an event occurring on DNA to the nucleus by protein modification and finally to the cytoplasm via NAD(+) depletion. It has also been proposed that the presence of negatively charged poly(ADP-ribose) at the site of DNA damage may play several roles in regulation of base excision repair, p53 functions, and apoptosis. This unit describes protocols for measuring the levels of poly(ADP-ribose) in cells using nonisotopic reagents and for identifying the poly(ADP-ribose) polymerase enzymes present in cells.