Hybrid epithelial-mesenchymal status of lung cancer dictates metastatic success through differential interaction with NK cells.

BACKGROUND: Epithelial to mesenchymal transition (EMT) endows cancer cells with pro-metastatic properties, which appear most effective when cells enter an intermediate hybrid (H) state, characterized by integrated mesenchymal (M) and epithelial (E) traits. The reasons for this advantage are poorly known and, especially, it is totally unexplored whether the interplay between H-cells and NK cells could have a role. Here we characterize the pro-metastatic mechanics of non-small cell lung cancer (NSCLC) H-cells and their subset of cancer-initiating cells (CICs), dissecting crucial interactions with NK cells. METHODS: Human lung cancer cell lines and sublines representative of E, M, or H states, assessed by proteomics, were analyzed in vivo for their tumor-forming and disseminating capabilities. Interactions with NK cells were investigated in vitro using migration assays, cytotoxic degranulation assays, and evaluation of CD133+ CICs modulation after coculture, and validated in vivo through NK cell neutralization assays. Correlation between EMT status, NK cell infiltration, and survival data, was evaluated in a cohort of surgically resected NSCLC cases (n=79). RESULTS: We demonstrated that H-cells, have limited dissemination capability but show the highest potential to initiate metastases in vivo. This property was related to their ability to escape NK cell surveillance. Mechanistically, H-cells expressed low levels of NK-attracting chemokines (CXCL1 and CXCL8), generating poorly infiltrated metastases. Accordingly, proteomics and GO enrichment analysis of E, H, M cell lines showed that the related secretory processes could change during EMT.Furthermore, H-CICs uniquely expressed high levels of the inhibitory ligand B7-H3, which protected H-CIC from NK cell-mediated clearance. In vivo neutralization assays confirmed that, indeed, the pro-metastatic properties of H-cells are poorly controlled by NK cells.Finally, the analysis of patients revealed that detection of hybrid phenotypes associated with low NK infiltration in NSCLC clinical specimens could identify a subset of patients with poor prognosis. CONCLUSIONS: Our study demonstrates that H-cells play a central role in the metastatic spread in NSCLC. Such pro-metastatic advantage of H-cells is supported by their altered interaction with NK cells and by the critical role of B7-H3 in preserving their H-CIC component, indicating B7-H3 as a potential target in combined NK-based therapies.

Mammalian PERIOD2 regulates H2A.Z incorporation in chromatin to orchestrate circadian negative feedback.

Mammalian circadian oscillators are built on a feedback loop in which the activity of the transcription factor CLOCK-BMAL1 is repressed by the PER-CRY complex. Here, we show that murine Per-/- fibroblasts display aberrant nucleosome occupancy around transcription start sites (TSSs) and at promoter-proximal and distal CTCF sites due to impaired histone H2A.Z deposition. Knocking out H2A.Z mimicked the Per null chromatin state and disrupted cellular rhythms. We found that endogenous mPER2 complexes retained CTCF as well as the specific H2A.Z-deposition chaperone YL1-a component of the ATP-dependent remodeler SRCAP and p400-TIP60 complex. While depleting YL1 or mutating chaperone-binding sites on H2A.Z lengthened the circadian period, H2A.Z deletion abrogated BMAL1 chromatin recruitment and promoted its proteasomal degradation. We propose that a PER2-mediated H2A.Z deposition pathway (1) compacts CLOCK-BMAL1 binding sites to establish negative feedback, (2) organizes circadian chromatin landscapes using CTCF and (3) bookmarks genomic loci for BMAL1 binding to impinge on the positive arm of the subsequent cycle.

MiR-486-5p Targets CD133+ Lung Cancer Stem Cells through the p85/AKT Pathway.

Despite improvements in therapies and screening strategies, lung cancer prognosis still remains dismal, especially for metastatic tumors. Cancer stem cells (CSCs) are endowed with properties such as chemoresistance, dissemination, and stem-like features, that make them one of the main causes of the poor survival rate of lung cancer patients. MicroRNAs (miRNAs), small molecules regulating gene expression, have a role in lung cancer development and progression. In particular, miR-486-5p is an onco-suppressor miRNA found to be down-modulated in the tumor tissue of lung cancer patients. In this study, we investigate the role of this miRNA in CD133+ lung CSCs and evaluate the therapeutic efficacy of coated cationic lipid-nanoparticles entrapping the miR-486-5p miRNA mimic (CCL-486) using lung cancer patient-derived xenograft (PDX) models. In vitro, miR-486-5p overexpression impaired the PI3K/Akt pathway and decreased lung cancer cell viability. Moreover, miR-486-5p overexpression induced apoptosis also in CD133+ CSCs, thus affecting the in vivo tumor-initiating properties of these cells. Finally, we demonstrated that in vivo CCL-486 treatment decreased CD133+ percentage and inhibited tumor growth in PDX models. In conclusion, we provided insights on the efficacy of a novel miRNA-based compound to hit CD133+ lung CSCs, setting the basis for new combined therapeutic strategies.

Differential glycosylation of collagen modulates lung cancer stem cell subsets through ß1 integrin-mediated interactions.

In lung cancer, CD133+ cells represent the subset of cancer stem cells (CSC) able to sustain tumor growth and metastatic dissemination. CSC function is tightly regulated by specialized niches composed of both stromal cells and extracellular matrix (ECM) proteins, mainly represented by collagen. The relevance of collagen glycosylation, a fundamental post-translational modification controlling several biological processes, in regulating tumor cell phenotype remains, however, largely unexplored. To investigate the bioactive effects of differential ECM glycosylation on lung cancer cells, we prepared collagen films functionalized with glucose (Glc-collagen) and galactose (Gal-collagen) exploiting a neoglycosylation approach based on a reductive amination of maltose and lactose with the amino residues of collagen lysines. We demonstrate that culturing of tumor cells on collagen determines a glycosylation-dependent positive selection of CSC and triggers their expansion/generation. The functional relevance of CD133+ CSC increase was validated in vivo, proving an augmented tumorigenic and metastatic potential. High expression of integrin ß1 in its active form is associated with an increased proficiency of tumor cells to sense signaling from glycosylated matrices (glyco-collagen) and to acquire stemness features. Accordingly, inhibition of integrin ß1 in tumor cells prevents CSC enrichment, suggesting that binding of integrin ß1 to Glc-collagen subtends CSC expansion/generation. We provide evidence suggesting that collagen glycosylation could play an essential role in modulating the creation of a niche favorable for the generation and selection/survival of lung CSC. Interfering with this crosstalk may represent an innovative therapeutic strategy for lung cancer treatment.

Circulating mir-320a promotes immunosuppressive macrophages M2 phenotype associated with lung cancer risk.

miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell-type specific expression pattern and topography of several miRNAs such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de-regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir-320a secreted by neutrophils of high-risk heavy-smokers promoted an M2-like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell-autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression.

Diagnostic role of circulating extracellular matrix-related proteins in non-small cell lung cancer.

BACKGROUND: Interactions between cancer cells and the surrounding microenvironment are crucial determinants of cancer progression. During this process, bi-directional communication among tumor cells and cancer associated fibroblasts (CAF) regulate extracellular matrix (ECM) deposition and remodeling. As a result of this dynamic process, soluble ECM proteins can be released into the bloodstream and may represent novel circulating biomarkers useful for cancer diagnosis. The aim of the present study was to measure the levels of three circulating ECM related proteins (COL11A1, COL10A1 and SPARC) in plasma samples of lung cancer patients and in healthy heavy-smokers controls and test whether such measurements have diagnostic or prognostic value. METHODS: Gene expression profiling of lung fibroblasts isolated from paired normal and cancer tissue of NSCLC patients was performed by gene expression microarrays. The prioritization of the candidates for the study of circulating proteins in plasma was based on the most differentially expressed genes in cancer associated fibroblasts. Soluble ECM proteins were assessed by western blot in the conditioned medium of lung fibroblasts and by ELISA assays in plasma samples. RESULTS: Plasma samples from lung cancer patients and healthy heavy-smokers controls were tested for levels of COL11A1 and COL10A1 (n = 57 each) and SPARC (n = 90 each). Higher plasma levels of COL10A1 were detected in patients (p ≤ 0.001), a difference that was driven specifically by females (p < 0.001). No difference in COL11A1 levels between patients and controls was found. SPARC levels were also higher in plasma patients than controls (p < 0.001) with good performance in discriminating the two groups (AUC = 0.744). No significant association was observed between plasma proteins levels and clinicopathological features or survival. CONCLUSION: Soluble factors related to proficient tumor-stroma cross-talk are detectable in plasma of primary lung cancer patients and may represent a valuable complementary diagnostic tool to discriminate lung cancer patients from healthy heavy-smokers individuals as shown for the SPARC protein.

MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner.

BACKGROUND: Fibroblasts are crucial mediators of tumor-stroma cross-talk through synthesis and remodeling of the extracellular matrix and production of multiple soluble factors. Nonetheless, little is still known about specific determinants of fibroblast pro-tumorigenic activity in lung cancer. Here, we aimed at understanding the role of miRNAs, which are often altered in stromal cells, in reprogramming fibroblasts towards a tumor-supporting phenotype. METHODS: We employed a co-culture-based high-throughput screening to identify specific miRNAs modulating the pro-tumorigenic potential of lung fibroblasts. Multiplex assays and ELISA were instrumental to study the effect of miRNAs on the secretome of both primary and immortalized lung fibroblasts from lung cancer patients and to evaluate plasmatic levels of HGF in heavy smokers. Direct mRNA targeting by miRNAs was investigated through dual-luciferase reporter assay and western blot. Finally, the pro-tumorigenic activity of fibroblasts and their conditioned media was tested by employing in vitro migration experiments and mouse xenografts. RESULTS: We identified miR-16 as a master regulator of fibroblast secretome and showed that its upregulation reduces HGF secretion by fibroblasts, impairing their capacity to promote cancer cell migration. This effect is due to a pleiotropic activity of miR-16 which prevents HGF expression through direct inhibition of FGFR-1 signaling and targeting of HGF mRNA. Mechanistically, miR-16 targets FGFR-1 downstream mediator MEK1, thus reducing ERK1/2 activation. Consistently, chemical or genetic inhibition of FGFR-1 mimics miR-16 activity and prevents HGF production. Of note, we report that primary fibroblast cell lines derived from lungs of heavy smokers express reduced miR-16 levels compared to those from lungs not exposed to smoke and that HGF concentration in heavy smokers' plasma correlates with levels of tobacco exposure. Finally, in vivo experiments confirmed that restoration of miR-16 expression in fibroblasts reduced their ability to promote tumor growth and that HGF plays a central role in the pro-tumorigenic activity of fibroblasts. CONCLUSIONS: Overall, these results uncover a central role for miR-16 in regulating HGF production by lung fibroblasts, thus affecting their pro-tumorigenic potential. Correlation between smoking exposure and miR-16 levels could provide novel clues regarding the formation of a tumor-proficient milieu during the early phases of lung cancer development.

Autologous fat tissue grafting improves pulmonary healing after laser metastasectomy.

BACKGROUND: Extensive clinical experience has demonstrated the potential usefulness of autologous fat tissue (AFT) graft in tissue reconstruction, repair or regeneration. In the present study, we evaluated the feasibility and safety of AFT in the repair of surgically injured lung surface. METHODS: Eighty consecutive procedures of pulmonary metastasectomy by laser precision resection, were performed in 66 patients between March 2010 and December 2012. In the first 20 procedures, AFT graft was applied on the wounded pulmonary surface without closure of parenchymal surface. The following 40 procedures were carried on without AFT (20 leaving the resection margins open and 20 closing the resection margins with a running suture). In the remaining 20 procedures, AFT was applied and the resection margins closed. The efficacy of this technique was evaluated by comparing the AFT group with the non-AFT group, with respect to prolonged alveolar air leakage (PAAL), time to drain removal, length of hospital stay, and patient survival at four years. RESULTS: The occurrence of PAAL was lower in the AFT group as compared to non-AFT group (17.5% versus 42.5%, p = 0.027), and median time to drain removal shorter (4 versus 6 days respectively, p = 0.016). Overall 4-year survival was 70% for AFT group, and 59% for non-AFT group (p = 0.34). CONCLUSIONS: This prospective cohort observational study demonstrated the feasibility and safety of AFT pulmonary grafting after laser metastasectomy. AFT graft improved pulmonary healing, by reducing the incidence and severity of PAAL. Moreover, there was no evidence of tumor promotion in the metastatic setting, with a similar overall survival at 4 years.

Early Effector T Lymphocytes Coexpress Multiple Inhibitory Receptors in Primary Non-Small Cell Lung Cancer.

Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed "early effector cells" (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non-small cell lung cancer. Cancer Res; 77(4); 851-61. ©2016 AACR.

Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung.

Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors.

Conversion to stem-cell state in response to microenvironmental cues is regulated by balance between epithelial and mesenchymal features in lung cancer cells.

Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non-CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFß1 to induce EMT and stimulate modulation of CD133(+) CICs. We show that response to TGFß1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5-2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E-cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer-associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem-cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity.

Complexity in the tumour microenvironment: Cancer associated fibroblast gene expression patterns identify both common and unique features of tumour-stroma crosstalk across cancer types.

Cancer is a complex disease, driven by the accumulation of several somatic aberrations but fostered by a two-way interaction between tumour cells and the surrounding microenvironment. Cancer associated fibroblasts (CAFs) represent one of the major players in tumour-stroma crosstalk. Recent in vitro and in vivo studies, often conducted by employing high throughput approaches, have started unravelling the key pathways involved in their functional effects. This review focus on open challenges in the study of CAF properties and function, highlighting at the same time the existence of common mechanisms as well as peculiarities in different cancer types (breast, prostate and lung cancer). Although still limited by current experimental models, which are unable to deal with the full level of complexity of the tumour microenvironment, a better understanding of these mechanisms may enable the identification of new biomarkers and therapeutic targets, to improve current strategies for cancer diagnosis and treatment.

Microenvironment-Modulated Metastatic CD133+/CXCR4+/EpCAM- Lung Cancer-Initiating Cells Sustain Tumor Dissemination and Correlate with Poor Prognosis.

Metastasis is the main reason for lung cancer-related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133(+)/CXCR4(+) cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133(+)/CXCR4(+) cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133(+)/CXCR4(+) with reduced expression of epithelial cell adhesion molecule (EpCAM(-)), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133(+)/CXCR4(+)/EpCAM(-) CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133(+)/CXCR4(+)/EpCAM(-) subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions.

Inactivation of both FHIT and p53 cooperate in deregulating proliferation-related pathways in lung cancer.

INTRODUCTION: FHIT and p53 are the two most commonly altered tumor suppressor genes in lung cancer, and their molecular status regulates sensitivity to anticancer drugs. Although their functions are independent, there is evidence that their pathways might be interconnected, but little is known at the molecular level. METHODS: Microarray profiling of FHIT-transduced lung cancer cells and modulation of FHIT levels by RNA interference in human bronchial cells were used to generate a signature of FHIT-regulated transcripts. Expression of these genes was evaluated by real-time polymerase chain reaction in 55 primary lung cancer samples characterized for FHIT and p53 expression by immunehistochemistry. RESULTS: A signature of FHIT-transcripts, particularly enriched in genes involved in cell cycle control, was identified. This signature showed overlap with p53-regulated genes, indicating possible crosstalk between these proteins. Consistently, transcriptional deregulation after FHIT modulation was higher in p53-negative cells. In primary lung cancers, inactivation of either gene was detected in 48 of 55 cases (87%) and both genes in 23 of 55 (42%) cases, confirming the central role of these pathways. Primary tumors with inactivation of both FHIT and p53 displayed the strongest deregulation of growth-related pathways with high levels of expression of CCNB1, BUB1, CDC6, TOP2A, MCM6, and CENPF. CONCLUSIONS: FHIT and p53 seem to rely on common mediators, and inactivation of both genes results in prominent deregulation of growth-related pathways in lung cancer cell lines and primary tumors. This reveals crosstalk between these proteins and suggests a possible distinctive phenotype for tumors with inactivation of both genes.

Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment.

The identification of lung tumor-initiating cells and associated markers may be useful for optimization of therapeutic approaches and for predictive and prognostic information in lung cancer patients. CD133, a surface glycoprotein linked to organ-specific stem cells, was described as a marker of cancer-initiating cells in different tumor types. Here, we report that a CD133+, epithelial-specific antigen-positive (CD133+ESA+) population is increased in primary nonsmall cell lung cancer (NSCLC) compared with normal lung tissue and has higher tumorigenic potential in SCID mice and expression of genes involved in stemness, adhesion, motility, and drug efflux than the CD133(-) counterpart. Cisplatin treatment of lung cancer cells in vitro resulted in enrichment of CD133+ fraction both after acute cytotoxic exposure and in cells with stable cisplatin-resistant phenotype. Subpopulations of CD133+ABCG2+ and CD133+CXCR4+ cells were spared by in vivo cisplatin treatment of lung tumor xenografts established from primary tumors. A tendency toward shorter progression-free survival was observed in CD133+ NSCLC patients treated with platinum-containing regimens. Our results indicate that chemoresistant populations with highly tumorigenic and stem-like features are present in lung tumors. The molecular features of these cells may provide the rationale for more specific therapeutic targeting and the definition of predictive factors in clinical management of this lethal disease.

Fragile histidine triad gene inactivation in lung cancer: the European Early Lung Cancer project.

RATIONALE: Fragile histidine triad (FHIT) is a tumor suppressor gene involved in the pathogenesis of lung cancer. OBJECTIVES: The purpose of this study was to investigate the different molecular alterations leading to the inactivation of FHIT gene function and to validate their use as biomarkers of risk for progression of the disease in patients belonging to the multicentric European study for the Early detection of Lung Cancer (EUELC) who were resected for early-stage lung tumors. METHODS: FHIT immunostaining was performed on 305 tumor samples. The methylation status of FHIT promoter was assessed by nested methylation-specific polymerase chain reaction (MSP-PCR) in 232 tumor and 225 normal lung samples of which a subset of 187 patients had available normal/tumor DNA pairs. Loss of heterozygosity (LOH) at the FHIT locus was analyzed in 202 informative cases by D3S1300 and D3S1234 microsatellite markers. MEASUREMENTS AND MAIN RESULTS: Lost or reduced FHIT expression was found in 36.7 and 75.7% of the tumor samples, respectively. Methylation of the FHIT promoter was found in 36.7% of tumor and 32.7% of normal lung samples, whereas LOH was detected in 61.9% of the tumors. A strong association with complete loss of FHIT expression was present when methylation and LOH were analyzed together (P = 0.0064). Loss of FHIT protein expression was significantly more frequent in squamous cell carcinoma histotype (P < 0.0001) and in smokers (P = 0.008). FHIT methylation in normal lung was associated with an increased risk of progressive disease (OR, 2.27; P = 0.0415). CONCLUSIONS: Our results indicate that different molecular mechanisms interplay to inactivate FHIT expression and support the proposition that FHIT methylation in normal lung tissue could represent a prognostic marker for progressive disease.

Plasma DNA quantification in lung cancer computed tomography screening: five-year results of a prospective study.

RATIONALE: Free circulating plasma DNA has emerged as a potential biomarker for early lung cancer detection. In a previous case-control study we have shown that high levels of plasma DNA are a strong risk factor for lung cancer. OBJECTIVES: To assess the diagnostic performance and prognostic value of plasma DNA levels in a cohort of 1,035 heavy smokers monitored by annual spiral computed tomography (CT) for 5 years. METHODS: Plasma DNA levels were determined through real-time quantitative PCR at baseline and at time of lung cancer diagnosis. Screening performance of the assay was calculated through the area under the receiver-operating characteristic curve (AUC-ROC). Kaplan-Meier analyses were computed for association with prognosis. MEASUREMENTS AND MAIN RESULTS: Median baseline concentration of plasma DNA was not different in individuals who developed CT-detected lung cancers in the 5-year period (n = 38) versus cancer-free control subjects (AUC-ROC, 0.496; P = 0.9330), and only slightly higher at the time of cancer diagnosis (AUC-ROC, 0.607; P = 0.0369). At surgery, plasma DNA was higher in tumors detected at baseline (AUC-ROC, 0.80; P < 0.0001) and in Stage II to IV tumors detected during the first 2 years of screening (AUC-ROC, 0.87; P < 0.0001). A longitudinal study of plasma DNA levels showed increased values approaching to lung cancer diagnosis (P = 0.0010). Higher plasma DNA was significantly associated with poorer 5-year survival (P = 0.0066). CONCLUSIONS: Baseline assessment of plasma DNA level does not improve the accuracy of lung cancer screening by spiral CT in heavy smokers. Higher levels of plasma DNA at surgery might represent a risk factor for aggressive disease.

Diadenosines as FHIT-ness instructors.

FHIT is a tumor suppressor gene that is frequently inactivated in human cancer. Although the Fhit protein is known to hydrolyze diadenosine triphosphate (Ap(3)A), this hydrolase activity is not required for Fhit-mediated oncosuppression. Indeed, the molecular mechanisms and the regulatory elements of Fhit oncosuppression are largely unknown. Here, we review physiological and pathological aspects of Fhit in the context of the Ap(n)A family of signaling molecules, as well as the involvement of Fhit in apoptosis and the cell cycle in cancer models. We also discuss recent findings of novel Fhit interactions that may lead to new hypotheses about biochemical mechanisms underlying the oncosuppressor activity of this gene.

Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays.

Analysis of molecular markers in biological fluids has been proposed as a tool for early detection and monitoring of cancer. Circulating plasma DNA concentrations have been found to be higher in cancer patients than in cancer-free control subjects, but little is known about the effect of specimen storage on plasma DNA concentrations. Here we investigated the impact of long-term storage of both plasma samples and purified plasma DNA on the reproducibility of plasma DNA quantification as determined using real-time polymerase chain reaction analysis. The analysis was performed on samples from a subset of 34 lung cancer patients and 28 matched control subjects selected from 200 subjects in our previously published case-control study and from 117 cancer-free smokers enrolled in a lung cancer screening program. Two samples of plasma and isolated DNA were assessed for each patient, with a median of 41 months between the first and second assessments for participants in the case-control study and 9 months for participants in the screening study. DNA levels declined substantially between the two assessments at an average rate of approximately 30% per year. These data provide valuable information for the rational planning of retrospective studies of banked series of biological samples, particularly if collected over a long period of time, as can occur in large clinical trials.