Early-stage non–small cell lung cancer (NSCLC) is potentially curable, however, many patients develop recurrent disease. Therefore, identification of biomarkers that can be used to predict patient’s risk of recurrence and survival is critical. Genetic polymorphisms or single-nucleotide polymorphisms (SNP) of DNA- and histone-modifying genes, particularly those of O6-methylguanine DNA-methyltransferase (MGMT), have been linked to an increased risk of lung cancer as well as treatment outcomes in other tumors.
We assessed the association of 165 SNPs in selected epigenetic enzyme genes, DNA methyltransferases, and methyl-CpG–binding proteins with cancer recurrence in 467 patients with stage I or II NSCLC treated with either surgery alone (N = 340) or surgery plus (neo)-adjuvant chemotherapy (N = 127).
We found several SNPs to be strongly correlated with tumor recurrence. We identified 10 SNPs that correlated with the outcome in patients treated with surgery alone but not in patients treated with surgery and adjuvant chemotherapy, which suggested that the addition of platinum-based chemotherapy could reverse the high genetic risk of recurrence. We also identified 10 SNPs that predicted the risk of recurrence in patients treated with surgery plus adjuvant chemotherapy but not in patients treated with surgery alone. The cumulative effect of these SNPs significantly predicted outcomes with P-values of 10−9and 10−6, respectively.
The first set of genotypes may be used as novel predictive biomarkers to identify patients with stage I NSCLC, who could benefit from adjuvant chemotherapy, and the second set of SNPs might predict response to adjuvant chemotherapy.
The optimal treatment for locally advanced superior sulcus tumors is not clear. The authors report long-term results of a trial examining the safety and efficacy of surgery followed by concurrent chemoradiation therapy for this disease.
Thirty-two patients with resectable or marginally resectable superior sulcus tumors at The University of Texas MD Anderson Cancer Center from 1994 to 2010 were enrolled in a prospective trial. Surgery involved segmentectomy or lobectomy with en bloc resection of the involved chest wall and complete nodal staging; radiation therapy (RT) began 14 to 42 days later to a dose of 60 grays (Gy) in 50 1.2-Gy fractions if surgical margins were negative or 64.8 Gy in 54 1.2-Gy fractions if margins were positive. Two cycles of etoposide (50 mg/ m2) and cisplatin (50 mg/m2) were given during RT, and another 3 cycles were given after RT. Eleven patients underwent prophylactic cranial irradiation (PCI).
The protocol completion rate was 78%. Gross total resection was accomplished in all 32 patients; 28% underwent R1 resection. Operative mortality was 0%. The most common surgical complication was postoperative pneumonia (25%). At a median follow-up time of 53.4 months (range, 2–154 months), the 2-year, 5-year, and 10-year rates of locoregional control were 84%, 76%, and 76%; distant metastasis-free survival, 52%, 48%, and 48%; disease-free survival, 49%, 45%, and 45%; and overall survival, 72%, 50%, and 45%, respectively. The brain was the most common site of distant failure (n =5), but no patient who received PCI experienced brain metastasis.
Surgery followed by postoperative chemoradiation is safe and effective for the treatment of marginally resectable superior sulcus tumors.
Pancoast tumor; lung neoplasms; adjuvant radiotherapy; adjuvant chemotherapy; chemoradiation
The CACNA2D2 gene, a new subunit of the Ca2+-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca2+ signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca2+ contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca2+ were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca2+ contents.
tumor suppressor genes; apoptosis; calcium channel proteins; human chromosome 3p21.3; lung cancer
A group of candidate tumor suppressor genes (designated CACNA2D2, PL6, 101F6, NPRL2, BLU, RASSF1, FUS1, HYAL2, and HYAL1) has been identified in a 120-kb critical tumor homozygous deletion region (found in lung and breast cancers) of human chromosome 3p21.3. We studied the effects of six of these 3p21.3 genes (101F6, NPRL2, BLU, FUS1, HYAL2, and HYAL1) on tumor cell proliferation and apoptosis in human lung cancer cells by recombinant adenovirus-mediated gene transfer in vitro and in vivo. We found that forced expression of wild-type FUS1, 101F6, and NPRL2 genes significantly inhibited tumor cell growth by induction of apoptosis and alteration of cell cycle processes in 3p21.3 120-kb region-deficient (homozygous) H1299 and A549 cells but not in the 3p21.3 120-kb region-heterozygous H358 and the normal human bronchial epithelial cells. Intratumoral injection of Ad-101F6, Ad-FUS1, Ad-NPRL2, and Ad-HYAL2 vectors or systemic administration of protamine-complexed vectors significantly suppressed growth of H1299 and A549 tumor xenografts and inhibited A549 experimental lung metastases in nu/nu mice. Together, our results, coupled with other studies demonstrating a tumor suppressor role for the RASSSF1A isoform, suggest that multiple contiguous genes in the 3p21.3 120-kb chromosomal region may exhibit tumor suppressor activity in vitro and in vivo.
Persistent pathologic mediastinal nodal involvement after induction chemotherapy and surgical resection is a negative prognostic factor for stage III-N2 non-small cell lung cancer patients. This population has high rates of local-regional failure and distant failure, yet the effectiveness of additional therapies is not clear. We assessed the role of consolidative therapies (postoperative radiation therapy and chemotherapy) for such patients.
In all, 179 patients with stage III-N2 non-small cell lung cancer at MD Anderson Cancer Center were treated with induction chemotherapy followed by surgery from 1998 through 2008; 61 patients in this cohort had persistent, pathologically confirmed, mediastinal nodal disease, and were treated with postoperative radiation therapy. Local-regional failure was defined as recurrence at the surgical site or lymph nodes (levels 1 to 14, including supraclavicular), or both. Overall survival was calculated using the Kaplan-Meier method, and survival outcomes were assessed by log rank tests. Univariate and multivariate Cox proportional hazards models were used to identify factors influencing local-regional failure, distant failure, and overall survival.
All patients received postoperative radiation therapy after surgery, but approximately 25% of the patients also received additional chemotherapy: 9 (15%) with concurrent chemotherapy, 4 (7%) received adjuvant sequential chemotherapy, and 2 (3%) received both. Multivariate analysis indicated that additional postoperative chemotherapy significantly reduced distant failure (hazard ratio 0.183, 95% confidence interval: 0.052 to 0.649, p = 0.009) and improved overall survival (hazard ratio 0.233, 95% confidence interval: 0.089 to 0.612, p = 0.003). However, additional postoperative chemotherapy had no affect on local-regional failure.
Aggressive consolidative therapies may improve outcomes for patients with persistent N2 disease after induction chemotherapy and surgery.
We evaluated the ability of histopathologic response criteria to predict overall survival (OS) and disease-free survival (DFS) in patients with surgically resected non-small cell lung cancer (NSCLC) treated with or without neoadjuvant chemotherapy.
Tissue specimens from 358 patients with NSCLC were evaluated by pathologists blinded to the patient treatment and outcome. The surgical specimens were reviewed for various histopathologic features in the tumor including percentage of residual viable tumor cells, necrosis, and fibrosis. The relationship between the histopathologic findings and OS was assessed.
The percentage of residual viable tumor cells and surgical pathologic stage were associated with OS and DFS in 192 patients with NSCLC receiving neoadjuvant chemotherapy in multivariate analysis (p = 0.005 and p = 0.01, respectively). There was no association of OS or DFS with percentage of viable tumor cells in 166 patients with NSCLC who did not receive neoadjuvant chemotherapy (p = 0.31 and p = 0.45, respectively). Long-term OS and DFS were significantly prolonged in patients who had ≤10% viable tumor compared with patients with >10% viable tumor cells (5 years OS, 85% versus 40%, p < 0.0001 and 5 years DFS, 78% versus 35%, p < 0.001).
The percentages of residual viable tumor cells predict OS and DFS in patients with resected NSCLC after neoadjuvant chemotherapy even when controlled for pathologic stage. Histopathologic assessment of resected specimens after neoadjuvant chemotherapy could potentially have a role in addition to pathologic stage in assessing prognosis, chemotherapy response, and the need for additional adjuvant therapies.
Lung cancer; Neoadjuvant chemotherapy; Histopathology
Clinical factors predicting pulmonary complications after lung resection have been well described, whereas the role of genetics is unknown. The vascular endothelial growth factor (VEGF) signaling pathway has been linked to acute lung injury. We hypothesized that genetic variations in this pathway may be associated with postoperative pulmonary complications after lung resection.
One hundred ninety-six single nucleotide polymorphisms (SNPs) in 17 genes in the VEGF pathway were genotyped in a discovery set of 264 patients and a replication set of 264 patients who underwent lobectomy for lung cancer. Multivariable analysis adjusting for baseline clinical factors was used to identify SNPs associated with pulmonary complications. Cumulative and classification and regression tree (CART) analyses were used to further stratify risk groups.
The overall number of pulmonary complications was 164/528 (31%). The effects of 6 SNPs were consistent in the discovery and replication sets (pooled p value < 0.05). The rs9319425 SNP in the VEGF receptor gene FLT1 resulted in a 1.50-fold increased risk (1.15–1.96; p = 0.003). A cumulative effect for the number of risk genotypes and complications was also evident (p < 0.01). Patients carrying 5 risk genotypes had a 5.76-fold increase in risk (2.73–12.16; p = 4.44 × 10−6). Regression tree analysis identified potential gene-gene interactions between FLT1:rs9319425 and RAF1:rs713178. The addition of the 6 SNPs to the clinical model increased the area under the receiver operating characteristic curve by 6.8%.
Genetic variations in the VEGF pathway are associated with risk of pulmonary complications after lobectomy. This may offer insight into the underlying biological mechanisms of pulmonary complications.
Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment and novel therapies are therefore in high demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug
The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter Insulinoma-associated 1 (INSM1). Therapeutic effect was evaluated in vitro in SCLC cell lines and in vivo in SCLC xenografted nude mice using the non-viral nanoparticle, DOTAP:Cholesterol for transgene delivery.
INSM1-YCD/5-FC and INSM1-YCD-YUPRT/5-FC therapy induced high cytotoxicity in a range of SCLC cell lines. The highest therapeutic effect was obtained from the YCD-YUPRT fusion gene strategy. No cytotoxicity was induced after treatment of cell lines of other origin than SCLC. In addition the INSM1-YCD-YUPRT/5-FC therapy was superior to an established suicide gene system consisting of the Herpes Simplex Virus Thymidine Kinase (HSVTK) gene and prodrug Ganciclovir (GCV). The superior effect was in part due to massive bystander cytotoxicity of YCD-YUPRT-produced toxins. Finally, INSM1-YCD-YUPRT/5-FC therapy induced significant tumor growth delay in SCLC xenografts compared to control treated xenografts.
The current study is the first to test cytosine deaminase-based suicide gene therapy for SCLC and the first to demonstrate an anti-tumor effect from the delivery of suicide gene therapeutics for SCLC in vivo.
gene therapy; transcriptional targeting; Insulinoma-associated 1 promoter; small cell lung cancer; suicide genes
Many cancers can become resistant to repeated administration of even the most effective therapeutic agents. In developing adenoviral mda-7/IL-24 (Ad-mda-7/IL-24) therapy for lung cancer, we have anticipated this potential clinical problem by attempting to identify the molecular mechanisms of Ad-mda7/IL-24 resistance in several Ad-mda7/IL-24-resistant lung cancer cell lines that we have developed. For the present study, we established four Ad-mda7-resistant cell lines by repeated selection of resistant clones of parental Ad-mda7-sensitive A549 cells: two lines (A549R1 and A549R2) resistant to both adenoviral vector and the mda-7 gene and two (A549R3 and A549R4) resistant to the therapeutic mda-7 gene only. As shown by western blot analysis of several known anti-apoptotic proteins, parental A549 and resistant A549R3 cells expressed similar levels of AKT and phosphorylated AKT (p-AKT), whereas resistant A549R3 and A549R4 cells expressed higher levels of bcl-2 and lower levels of bcl-xL than did their parental cells. As shown by flow-cytometric analysis, treating resistant A549R3 and A549R4 cells with a combination of Ad-mda7 and 17-allylamino-17-demethoxygeldanamycin (17AAG) (50 nM) for 48 hours enhanced apoptosis. Together, these in vitro findings indicate that an antiapoptotic mechanism may underlie Ad-mda7 resistance and that such resistance can be overcome by addition of 17AAG. Further investigations along these lines are warranted.
resistant cell lines; apoptosis; MDA-7; adenovirus; gene therapy
Retinoids have shown antiproliferative and chemopreventive activity. We analyzed data from a randomized, placebo-controlled chemoprevention trial to determine whether a 3-month treatment with either 9-cis-retinoic acid (RA) or 13-cis-RA and α-tocopherol reduced Ki-67, a proliferation biomarker, in the bronchial epithelium.
Former smokers (n = 225) were randomly assigned to receive 3 months of daily oral 9-cis-RA (100 mg), 13-cis-RA (1 mg/kg) and α-tocopherol (1200 IU), or placebo. Bronchoscopic biopsy specimens obtained before and after treatment were immunohistochemically assessed for changes in the Ki-67 proliferative index (i.e., percentage of cells with Ki-67–positive nuclear staining) in the basal and parabasal layers of the bronchial epithelium. Per-subject and per–biopsy site analyses were conducted. Multicovariable analyses, including a mixed-effects model and a generalized estimating equations model, were used to investigate the treatment effect (Ki-67 labeling index and percentage of bronchial epithelial biopsy sites with a Ki-67 index ≥ 5%) with adjustment for multiple covariates, such as smoking history and metaplasia. Coefficient estimates and 95% confidence intervals (CIs) were obtained from the models. All statistical tests were two-sided.
In per-subject analyses, Ki-67 labeling in the basal layer was not changed by any treatment; the percentage of subjects with a high Ki-67 labeling in the parabasal layer dropped statistically significantly after treatment with 13-cis-RA and α-tocopherol treatment (P = .04) compared with placebo, but the drop was not statistically significant after 9-cis-RA treatment (P = .17). A similar effect was observed in the parabasal layer in a per-site analysis; the percentage of sites with high Ki-67 labeling dropped statistically significantly after 9-cis-RA treatment (coefficient estimate = −0.72, 95% CI = −1.24 to −0.20; P = .007) compared with placebo, and after 13-cis-RA and α-tocopherol treatment (coefficient estimate = −0.66, 95% CI = −1.15 to −0.17; P = .008).
In per-subject analyses, treatment with 13-cis-RA and α-tocopherol, compared with placebo, was statistically significantly associated with reduced bronchial epithelial cell proliferation; treatment with 9-cis-RA was not. In per-site analyses, statistically significant associations were obtained with both treatments.
Stereotactic ablative radiotherapy (SABR) can achieve excellent local control rates in early-stage non-small cell lung cancer (NSCLC) and has emerged as a standard treatment option for patients who cannot undergo surgery or those with isolated recurrences. However, factors that may predict toxicity or survival are largely unknown. We sought here to identify predictors of survival and pneumonitis after SABR for NSCLC in a relatively large single-institution series.
Subjects were 130 patients with stage I NSCLC treated with four-dimensional computed tomography (4D CT) –planned, on-board volumetric image–guided SABR to 50 Gy in 4 fractions. Disease was staged by positron emission tomography/computed tomography (PET/CT) and scans were obtained again at the second follow-up after SABR.
At a median follow-up time of 26 months, the 2-year local control rate was 98.5%. The median overall survival (OS) time was 60 months, and OS rates were 93.0% at 1 year, 78.2% at 2 years, and 65.3% at 3 years. No patient experienced grade 4–5 toxicity; 15 had radiation pneumonitis (12 [9.3%] grade 2 and 3 [2.3%] grade 3). Performance status, standardized uptake value (SUV)max on staging PET/CT, tumor histology, and disease operability were associated with OS on univariate analysis, but only staging SUVmax was independently predictive on multivariate analysis (P = 0.034). Dosimetric factors were associated with radiation pneumonitis on univariate analysis, but only mean ipsilateral lung dose ≥9.14 Gy was significant on multivariate analysis (P = 0.005).
OS and radiation pneumonitis after SABR for stage I NSCLC can be predicted by staging PET SUVmax and ipsilateral mean lung dose, respectively.
Stereotactic body radiotherapy; Stereotactic ablative radiotherapy; Non-small cell lung cancer; F-fluorodeoxyglucose positron emission tomography; Toxicity; Predictive factors
The requirement of frozen tissues for microarray experiments limits the clinical usage of genome-wide expression profiling using microarray technology. The goal of this study is to test the feasibility of developing lung cancer prognosis gene signatures using genome-wide expression profiling of formalin-fixed paraffin-embedded (FFPE) samples, which are widely available and provide a valuable rich source for studying the association of molecular changes in cancer and associated clinical outcomes.
We randomly selected 100 Non-Small-Cell lung cancer (NSCLC) FFPE samples with annotated clinical information from the UT-Lung SPORE Tissue Bank. We micro dissected tumor area from FFPE specimens, and used Affymetrix U133 plus 2.0 arrays to attain gene expression data. After strict quality control and analysis procedures, a supervised principal component analysis was used to develop a robust prognosis signature for NSCLC. Three independent published microarray data sets were used to validate the prognosis model.
This study demonstrated that the robust gene signature derived from genome-wide expression profiling of FFPE samples is strongly associated with lung cancer clinical outcomes, can be used to refine the prognosis for stage I lung cancer patients and the prognostic signature is independent of clinical variables. This signature was validated in several independent studies and was refined to a 59-gene lung cancer prognosis signature.
We conclude that genome-wide profiling of FFPE lung cancer samples can identify a set of genes whose expression level provides prognostic information across different platforms and studies, which will allow its application in clinical settings.
Lung Cancer Prognosis; Gene Expression Signature; Formalin Fixed Paraffin Embedded Samples
Activating enhancer-binding protein-2β (AP2β) is a transcription factor involved in apoptosis. The purpose of the current study was to assess the cellular location and level of AP2β in Non-Small Cell Lung Cancer (NSCLC) and normal lung tissue and investigate whether the level and localization of AP2β expression is predictive of overall survival in patients with stage I NSCLC.
We performed immunohistochemical analysis of tissue microarrays (TMAs) prepared from stage I NSCLC specimens with adjacent normal lung tissue from two independent sets of patients who underwent lung resection with curative intent at our institution. AP2β intensity was assessed in TMAs, and AP2β staining patterns were classified as either diffuseor nucleolar in the TMAs. AP2β intensity and localization were analyzed for correlation with patients' survival.
Immunohistochemical analysis of TMAs showed that the intensity of AP2β immunohistochemical staining did not correlate with overall survival. When location of AP2β was analyzed in TMAs, all of the normal lung tissue had diffuse pattern of AP2β. In the first set of NSCLC, patients with nucleolar pattern had a significantly lower 5-year survival rate than patients with diffuse pattern (67% vs. 100%; P = 0.004); this finding was confirmed in the second set (64% vs. 91%; P = 0.02). Multivariate analysis revealed that nucleolar pattern was an independent predictor of poor overall survival in both sets.
The AP2β which is located in the nucleoplasm in normal lung tissue is found in either nucleoplasm or nucleoli in NSCLC. The patients with AP2β in the nucleoli had poor survival compared to patients with AP2β in the cytoplasm.
Lung cancer biology; survival analysis
AZD6244 is a small molecule inhibitor of the MEK kinase pathway currently in clinical trials. However, the mechanisms mediating intrinsic resistance to MEK inhibition are not fully characterized. To define molecular mechanisms of MEK inhibitor resistance, we analyzed responses of 38 lung cancer cell lines following AZD6244 treatment and their genome-wide gene expression profiles and identified a panel of genes correlated with sensitivity or resistance to AZD6244 treatment. In particular, Ingenuity pathway analysis revealed that activation of the STAT3 pathway was associated with MEK inhibitor resistance. Inhibition of this pathway by JSI-124, a STAT3-specific small molecule inhibitor, or with STAT3-specific siRNA sensitized lung cancer cells to AZD6244 and induced apoptosis. Moreover, combining a STAT3 inhibitor with AZD6244 induced expression of BIM and polyADP-ribose polymerase (PARP) cleavage, whereas activation of the STAT3 pathway inhibited BIM expression and elicited resistance to MEK inhibitors. We found that the STAT3-regulated microRNA miR-17 played a critical role in MEK inhibitor resistance, such that miR-17 inhibition sensitized resistant cells to AZD6244 by inducing BIM and PARP cleavage. Together, these results indicated that STAT3-mediated overexpression of miR-17 blocked BIM expression and caused resistance to AZD6244. Our findings suggest novel approaches to overcome resistance to MEK inhibitors by combining AZD6244 with STAT3 or miR-17 inhibitors.
Gene expression profiling; MEK inhibitor resistance; AZD6244; STAT3 pathway; miR-17
FUS1, also known as tumor suppressor candidate 2 (TUSC2), is a tumor suppressor gene located in the human chromosome 3p21.3 region. FUS1 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous FUS1 protein could be detected in a majority of lung cancer cell lines and small cell and non-small cell lung tumor tissues. However, mechanisms regulating FUS1 protein expression and its inactivation in primary lung cancer cells are largely unknown. In this study, we investigated the role of the 5′- and 3′-untranslated regions (UTRs) of the FUS1 gene transcript in the regulation of FUS1 protein expression. We identified RNA sequence elements in FUS1 UTRs that regulate FUS1 protein expression. We found that two small upstream open-reading frames in the 5 UTR of FUS1 mRNA could inhibit the translational initiation of FUS1 protein by interfering with the “scanning” of the ribosome initiation complexes. Several secondary RNA structural elements/motifs on the 3′UTR of FUS1 also exhibited a significant inhibitory effect on FUS1 protein expression. The 3′UTR-mediated regulatory effect on FUS1 protein expression was also differentially detected in normal lung epithelial and fibroblast cells compared with lung cancer cells. Our results provide new insight into the molecular mechanisms involved in the regulation of FUS1 expression.
FUS1/TUSC2; Tumor suppressor gene; Lung cancer; untranslated region (UTR); upstream open reading frame (uORF); expression regulation
Detection of lung cancer by sputum cytology has low sensitivity but is noninvasive and, if improved, could be a powerful tool for early lung cancer detection. To evaluate whether the accuracy of diagnosing lung cancer by evaluating sputa for cytologic atypia and genetic abnormalities is greater than that of conventional cytology alone, automated scoring of genetic abnormalities for 3p22.1 and 10q22.3 (SP-A) by fluorescence in situ hybridization (FISH) and conventional cytology was done on sputa from 35 subjects with lung cancer, 25 high-risk smokers, and 6 healthy control subjects. Multivariate analysis was performed to select variables that most accurately predicted lung cancer. A model of probability for the presence of lung cancer was derived for each subject. Cells exfoliated from patients with lung cancer contained genetic aberrations and cytologic atypias at significantly higher levels than in those from control subjects. When combined with cytologic atypia, a model of risk for lung cancer was derived that had 74% sensitivity and 82% specificity to predict the presence of lung cancer, whereas conventional cytology achieved only 37% sensitivity and 87% specificity. For diagnosing lung cancer in sputum, a combination of molecular and cytologic variables was superior to using conventional cytology alone.
surfactant protein A gene; 3p22.1; FISH; cytology; field cancerization effect; sputum
The identification of key pathways dysregulated in non-small cell lung cancer (NSCLC) is an important step toward understanding lung pathogenesis and developing new therapeutic approaches.
Toward this goal, reverse-phase protein lysate arrays (RPPA) were used to compare signaling pathways between NSCLC tumors and paired normal lung tissue from 46 patients and assess their association with clinical outcome.
After RPPA quantification of 63 proteins and phosphoproteins, tissue pairs were randomized to a training set (n = 25 pairs) and test set (n = 21 pairs). In the training set, 15 protein markers were differentially expressed between tumors and normal lung (p ≤ 0.01), including markers in the PI3K/AKT and p38 MAPK signaling pathways (e.g., p70S6K, S6, p38, and phospho-p38), as well as caveolin-1 and β-catenin. A four-protein signature (p70S6K, cyclin B1, pSrc(Y527), and caveolin-1) independent of histology classified specimens as tumor versus normal with a predicted accuracy of 83%, sensitivity of 67%, and specificity of 100%. The signature was validated in the test set, correctly classifying all normal tissues and 14 of 21 tumor tissues. RPPA results were confirmed by immunohistochemistry for caveolin-1 and p70S6K. In tumors from patients with resected NSCLC, expression of proteins in the energy-sensing AMPK pathway (pLKB1, AMPK, p-Acetyl-CoA, pTSC2), adhesion, EGFR, and Rb signaling pathways was inversely associated with NSCLC recurrence.
These data provide evidence for dysregulation of several pathways including those involving energy sensing and adhesion that are potentially associated with NSCLC pathogenesis and disease recurrence.
NSCLC; Proteomics; Recurrence; AMPK; Adhesion
The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in combination with the mutant p53-reactivating molecule, PRIMA-1Met/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1Met/APR-246, a synergistic cell growth inhibition was achieved.
Tumor suppressor gene; SCLC; FHIT; p53
FUS1 is a novel tumor suppressor gene identified in the human chromosome 3p21.3 region where allele losses and genetic alterations occur early and frequently for many human cancers. Expression of FUS1 protein is absent or reduced in the majority of lung cancers and premalignant lung lesions. Restoration of wt-FUS1 function in 3p21.3-deficient non-small cell lung carcinoma cells significantly inhibits tumor cell growth by induction of apoptosis and alteration of cell cycle kinetics. Here we present recent findings indicating that FUS1 induces apoptosis through the activation of the intrinsic mitochondrial-dependent and Apaf-1-associated pathways and inhibits the function of protein tyrosine kinases including EGFR, PDGFR, AKT, c-Abl, and c-Kit. Intravenous administration of a nanoparticle encapsulated FUS1 expression plasmid effectively delivers FUS1 to distant tumor sites and mediates an antitumor effect in orthotopic human lung cancer xenograft models. This approach is the rationale for an ongoing FUS1-nanoparticle-mediated gene delivery clinical trial for the treatment of lung cancer.
Tumor suppressor gene; FUS1; Signaling pathway; Lung cancer
Our objective was to study the feasibility of detecting chromosomal deletions at 3p22.1 and 10q22.3 by fluorescent in situ hybridization (FISH) and to examine their distribution in different areas of the airway in patients with non-small cell lung cancer.
Brush biopsies from the mainstem bronchus on the normal side contralateral to the tumor (NBB) and mainstem bronchus on the tumor side (TBB) were obtained from 122 patients who underwent surgical resection. Touch preparations from the tumor (TTP), normal lung parenchyma, and bronchi adjacent to the tumor were also obtained. Two FISH assays using probes complementary to 3p22.1 and 10q22.3 were used to detect deletions.
NBB showed a relatively low deletion rate of 3p22.1 and 10q22.3 compared with TTP (p < 0.0001). TBB showed a significantly higher rate of deletions compared with NBB but lower than TTP from the tumor (p < 0.05) for both 3p22.1 and 10q22.3. A significantly higher deletion rate was seen at TTP compared with normal lung parenchyma at both the 3p22.1 and 10 q22.3 (p < 0.0001). Correlations were seen between the deletion rates of TTP and TBB at 3p22.1 (ρ = 0.61, p < 0.0001) and between TTP and bronchi adjacent to the tumor at 10q22.3 (ρ = 0.64, p < 0.0001).
Deletions of the 3p22.1 and 10q22.3 regions can be reliably detected by FISH. As one progresses from the contralateral normal bronchus to the bronchus on the side of tumor and the tumor itself, the percentage of chromosomal deletions increases in a statistically significant fashion. This suggests that, FISH analysis of bronchoscopic brushes may be useful for identifying patients at high risk for developing non-small cell lung cancer.
Lung; Cancer; Non-small cell; Screening; Fluorescent in situ hybridization; Bronchial brushes
Protein–protein interactions are key elements in the assembly of cellular regulatory and signaling protein complexes that integrate and transmit signals and information in controlling and regulating various cellular processes and functions. Many conventional methods of studying protein–protein interaction, such as the immuno-precipitation and immuno-blotting assay and the affinity-column pull-down and chromatographic analysis, are very time-consuming and labor intensive and lack accuracy and sensitivity. We have developed a simple, rapid, and sensitive assay using a ProteinChip array and SELDI-TOF mass spectrometry to analyze protein–protein interactions and map the crucial elements that are directly involved in these interactions. First, a purified “bait” protein or a synthetic peptide of interest is immobilized onto the pre-activated surface of a PS10 or PS20 ProteinChip and the unoccupied surfaces on the chip are protected by application of a layer ethanolamine to prevent them from binding to other non-interactive proteins. Then, the target-containing cellular protein lysate or synthetic peptide containing the predicted amino acid sequence of protein-interaction motif is applied to the protected array with immobilized bait protein/ peptide. The nonspecific proteins/peptides are washed off under various stringent conditions and only the proteins specifically interacting with the bait protein/peptide remain on the chip. Last, the captured interacting protein/peptide complexes are then analyzed by SELDI-TOF mass spectrometry and their identities are confirmed by their predicted distinctive masses. This method can be used to unambiguously detect the specific protein–protein interaction of known proteins/peptides, to easily identify potential cellular targets of proteins of interest, and to accurately analyze and map the structural elements of a given protein and its target proteins using synthetic peptides with the predicted potential protein interaction motifs.
SELDI-TOF-MS; ProteinChip; Peptide interactions; SEND-ID; Fus1; Apaf1
Protein phosphorylation is a dynamic post-translational modification that plays a critical role in the regulation of a wide spectrum of biological events and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis. Determination of the sites and magnitudes of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. A high throughput analysis of phosphorylation of proteins would provide a simple, logical, and useful tool for a functional dissection and prediction of biological functions and signaling pathways in association with these important molecular events. We have developed a functional proteomics technique using the ProteinChip array-based SELDI-TOF-MS analysis for high throughput profiling of phosphoproteins/phosphopeptides in human serum for the early detection and diagnosis as well as for the molecular staging of human cancer. The methodology and experimental approach consists of five steps: (1) generation of a total peptide pool of serum proteins by a global trypsin digestion; (2) rapid isolation of phosphopeptides from the total serum peptide pool by an affinity selection, purification, and enrichment using a novel automated micro-bioprocessing system with phospho-antibody-conjugated paramagnetic beads and a hybrid magnet plate; (3) high throughput phosphopeptide analysis on ProteinChip arrays by automated SELDI-TOF-MS; and (4) bioinformatics and statistical methods for data analysis. This method with appropriate modifications may be equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins and for selectively isolating, profiling, and identifying phosphopeptides present in a highly complex phosphor-peptide mixture prepared from various human specimens such as cells, tissue samples, and serum and other body fluids.
Phosphoprotein; Phosphopeptide; Phosphoproteome; High throughput Phospoprotein/Peptide Profiling; ProteinChip Arrays; SELDI-TOF-MS
Interindividual variation in genetic background may influence the response to chemotherapy and overall survival for patients with advanced-stage non–small cell lung cancer (NSCLC).
To identify genetic variants associated with poor overall survival in these patients, we conducted a genome-wide scan of 307 260 single-nucleotide polymorphisms (SNPs) in 327 advanced-stage NSCLC patients who received platinum-based chemotherapy with or without radiation at the University of Texas MD Anderson Cancer Center (the discovery population). A fast-track replication was performed for 315 patients from the Mayo Clinic followed by a second validation at the University of Pittsburgh in 420 patients enrolled in the Spanish Lung Cancer Group PLATAX clinical trial. A pooled analysis combining the Mayo Clinic and PLATAX populations or all three populations was also used to validate the results. We assessed the association of each SNP with overall survival by multivariable Cox proportional hazard regression analysis. All statistical tests were two-sided.
SNP rs1878022 in the chemokine-like receptor 1 (CMKLR1) was statistically significantly associated with poor overall survival in the MD Anderson discovery population (hazard ratio [HR] of death = 1.59, 95% confidence interval [CI] = 1.32 to 1.92, P = 1.42 × 10−6), in the PLATAX clinical trial (HR of death = 1.23, 95% CI = 1.00 to 1.51, P = .05), in the pooled Mayo Clinic and PLATAX validation (HR of death = 1.22, 95% CI = 1.06 to 1.40, P = .005), and in pooled analysis of all three populations (HR of death = 1.33, 95% CI = 1.19 to 1.48, P = 5.13 × 10−7). Carrying a variant genotype of rs10937823 was associated with decreased overall survival (HR of death = 1.82, 95% CI = 1.42 to 2.33, P = 1.73 × 10−6) in the pooled MD Anderson and Mayo Clinic populations but not in the PLATAX trial patient population (HR of death = 0.96, 95% CI = 0.69 to 1.35).
These results have the potential to contribute to the future development of personalized chemotherapy treatments for individual NSCLC patients.
Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.
Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.
Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).
DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).
Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues.
Methodology/ Principal Findings
We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA) assay. The results showed that 18 molecules were significantly different (p<0.05) by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery.
Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers.