Differential modulation of PI3K/Akt/mTOR activity by EGFR inhibitors: A rationale for co-targeting EGFR and PI3K in cisplatin-resistant HNSCC.

PURPOSE: To find a new strategy to treat cisplatin-resistant head and neck squamous cell carcinoma (HNSCC), we investigated the effects of EGFR inhibitors on the PI3K/Akt/mTOR pathway and determined the efficacy of EGFR inhibitors in combination with PI3K inhibitors to suppress cell proliferation in cisplatin-resistant-HNSCC. METHODS: The cisplatin-resistant HNSCC cell lines were treated with four FDA approved EGFR inhibitors, which included Gefitinb or Erlotinib alone, or in combination with the pan-PI3K inhibitor, BKM120. Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell proliferation was examined by MTS assay. Apoptosis was analyzed by flow cytometry. RESULTS: Cisplatin-resistant HNSCC cells were also resistant to EGFR inhibitors. However, a combination of EGFR inhibitors with PI3K inhibitor BKM120 dramatically improved the efficacy of EGFR inhibitors to inhibit cell proliferation and induce apoptosis. Furthermore, treatment with EGFR inhibitors differentially affected the phosphorylation of Akt and mTOR, which included partial inhibition, no inhibition, and induction. A combination of EGFR inhibitors and BKM120 completely blocked phosphorylation of EGFR, Akt, and S6K (an mTOR target). CONCLUSION: Our data provided a rationale for EGFR inhibitors in combination with PI3K inhibitors to treat cisplatin-resistant HNSCC.

Circulating Bacterial DNA as Plasma Biomarkers for Lung Cancer Early Detection.

Lung cancer is a leading cause of cancer deaths and early diagnosis can significantly improve outcomes. Pathogenic bacteria have been shown to play a role in tumorigenesis and its analysis provides a new approach for cancer diagnosis. To evaluate the potential of bacteria as plasma biomarkers for early lung cancer detection, we analyzed eight lung-cancer-related bacterial genera in 58 lung cancer patients and 58 controls using ddPCR. Our results showed that five genera had higher DNA abundance in lung tumor tissues compared with normal tissues. Three of these genera (Selenomonas, Streptococcus, and Veillonella) displayed consistent changes in plasma, with higher DNA abundance in lung cancer patients compared with controls. When used as a panel, these three bacterial genera had a sensitivity of 75% and specificity of 78% for lung cancer detection, regardless of stage or histology. The performance of this biomarker panel was confirmed in an independent cohort of 93 lung cancer cases and 93 controls. Thus, circulating bacterial DNA has the potential to be used as plasma biomarkers for early lung cancer detection.

Simultaneously targeting ErbB family kinases and PI3K in HPV-positive head and neck squamous cell carcinoma.

OBJECTIVES: To identify the most effective PI3K and EGFR inhibitors in HPV-positive head and neck squamous cell carcinoma (HNSCC) and investigate the efficacy of a combination of an ErbB family kinase inhibitor and a PI3K inhibitor to inhibit cell proliferation of HPV-positive HNSCC. MATERIALS AND METHOD: HPV-positive HNSCC cell lines were treated with the FDA approved ErbB kinase inhibitor, Afatinib or FDA-approved PI3K inhibitor, Copanlisib, alone or in combination, and phosphorylation and total protein levels of cells were assessed by Western blot analysis.Cell proliferation and apoptosis were examined by MTS assay, flow cytometry, and Western blots, respectively. RESULTS: Copanlisib more effectively inhibited cell proliferation in comparison to other PI3K inhibitors tested. HPV-positive HNSCC cells differentially responded to cisplatin, Afatinib, or Copanlisib. The combination of Afatinib and Copanlisib more effectively suppressed cell proliferation and induced apoptosis compared to either treatment alone. Mechanistically, the combination of Afatinib and Copanlisib completely blocked phosphorylation of EGFR, HER2, HER3, and Akt as well as significantly decreased the HPV E7 expression compared to either treatment alone. CONCLUSION: Afatinib and Copanlisib more effectively suppress cell proliferation and survival of HPV-positive HNSCC in comparison to either treatment alone.

Targeting Wee1 kinase to suppress proliferation and survival of cisplatin-resistant head and neck squamous cell carcinoma.

PURPOSE: We investigated the role of Wee1 kinase in cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines and determined the efficacy of either Wee1 inhibitor, AZD1775 alone, or in combination with cisplatin, on cisplatin-resistant HNSCC inhibition. METHODS: Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell viability and apoptosis were examined by MTS assay and flow cytometry, respectively. RESULTS: Wee1 kinase protein expression levels in five cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin-sensitive partners. Importantly, Wee1 knockdown inhibited cell proliferation and re-sensitized cells to cisplatin treatment. Interestingly, previous studies have also shown that Wee1 inhibitor AZD1775 synergizes with cisplatin to suppress cell proliferation of cisplatin-sensitive HNSCC. We found that AZD1775 inhibited both cisplatin-sensitive and resistant HNSCC with similar IC50 values, which suggested that AZD1775 could overcome cisplatin resistance in cisplatin-resistant HNSCC. Mechanistically, AZD1775 and cisplatin cooperatively induced DNA damage and apoptosis. CONCLUSION: Wee1 inhibitor, AZD1775, and cisplatin coordinately suppressed proliferation and survival of HNSCC.

Inhibition of IKKß/NF-κB signaling pathway to improve Dasatinib efficacy in suppression of cisplatin-resistant head and neck squamous cell carcinoma.

Proto-oncogene tyrosine-protein kinase Src plays an important role in Head and Neck Squamous Cell Carcinoma (HNSCC). However, the FDA-approved SRC inhibitor Dasatinib shows very limited efficacy in HNSCC clinical trials, even though Dasatinib can completely inhibit SRC in the laboratory setting. These results suggest that SRC inhibition can cause compensatory up-regulation and/or activation of other survival pathways, which suggests that co-targeting of SRC and the potential signaling pathways may improve the Dasatinib efficacy. In this study, we investigated the role of IKKß/NF-κB in regulation of the sensitivity of cisplatin-resistant HNSCC to Dasatinib. Additionally, we wished to determine whether inhibition of the IKKß/NF-κB signaling pathway could enhance Dasatinib efficacy to inhibit cisplatin-resistant HNSCC without the use of cisplatin. Previous studies have shown that ETS-1 is a crucial SRC effector protein that regulates cancer cell proliferation, anti-apoptosis, and metastasis. We found that SRC kinase inhibition by Dasatinib decreased ETS-1 expression but caused elevation of IKKß/NF-κB signaling in multiple cisplatin-resistant HNSCC. Interestingly, inhibition of IKKß/NF-κB by CmpdA (Bay65-1942), a recently identified IKKß inhibitor, also led to a decrease in ETS-1 levels. Moreover, the knockdown of IKK, but not NF-κB, dramatically decreased ETS-1 expression. In addition, IKKß and ETS-1 interacted in cisplatin-resistant HNSCC. These data demonstrated cross-talk between SRC and IKK to regulate NF-κB and ETS-1. Furthermore, we found that simultaneous inhibition of SRC and IKKß through a Dasatinib and CmpdA combination synergistically inhibited NF-κB activation and ETS-1expression, suppressed cell proliferation, and induced apoptosis. Taken together, our data indicate that SRC and IKKß play crucial roles in cisplatin-resistant HNSCCC and co-targeting SRC and IKKß could be an effective strategy to treat cisplatin-resistant HNSCC.

Concurrent inhibition of ErbB family and MEK/ERK kinases to suppress non-small cell lung cancer proliferation.

Lung cancer ranks as the most common cancer and leading cause of cancer-related deaths worldwide. Of all lung cancer types, non-small cell lung cancer (NSCLC) accounts for 85 percent of all cases. The high mortality of NSCLC occurs mainly because of poor prognosis in patients with recurrent and metastatic cancer. Cisplatin-containing chemotherapy is the first option to treat recurrent and metastatic NSCLC. Additionally, targeted therapy plays an important role to prolong life in patients. Currently, EGFR inhibitors are the most important targeted anti-cancer drugs for patients with EGFR mutations in the clinical setting. Another important kinase inhibitor for targeted therapy is the MEK inhibitor, Trametinib, which is often used for patients with BRAF mutation or MEK/ERK activation in the tumors. In this study, we determined whether a combination of the pan-ErbB kinase inhibitor, Afatinib, and MEK inhibitor, Trametinib, could more effectively inhibit NSCLC cell proliferation when compared to either single treatment. We found that Afatinib inhibited phosphorylation of EGFR, HER2, HER3, and HER4, as well as Akt, whereas it elevated ERK phosphorylation. Conversely, Trametinib treatment led to ERK inhibition, but induced Akt phosphorylation. However, the combination of Afatinib and Trametinib inhibited all of the above-mentioned signaling pathways and synergistically suppressed cell proliferation. Our data indicate that co-targeting of ErbB family and MEK/ERK pathways through a combination of Afatinib and Trametinib could be a potential effective strategy to treat NSCLC.

Suppression of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma through IKKß inhibition.

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKß in regulation of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We showed that cisplatin-resistant HNSCC cells have a stronger ability to migrate and invade, as well as display higher IKKß/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKß, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKß inhibitor, CmpdA, also inhibited cell migration and invasion. Previous studies have already shown that N-Cadherin, an epithelial-mesenchymal transition (EMT) marker, and IL-6, a pro-inflammatory cytokine, play important roles in regulation of HNSCC migration, invasion, and metastasis. We found that cisplatin-resistant HNSCC expressed higher levels of N-Cadherin and IL-6, which were significantly inhibited by CmpdA. More importantly, we showed that CmpdA treatment dramatically abated cisplatin-resistant HNSCC cell metastasis to lungs in a mouse model. Our data demonstrated the crucial role of IKKß in control of migration, invasion, and metastasis, and implicated that targeting IKKß may be a potential therapy for cisplatin-resistant metastatic HNSCC.

MicroRNA-based biomarkers for diagnosis of non-small cell lung cancer (NSCLC).

BACKGROUND: The development of biomarkers for the early detection of non-small cell lung cancer (NSCLC) is clinically important. We have developed miRNA biomarkers in sputum and plasma, respectively, for NSCLC. Herein, we evaluate whether integrated analysis of the miRNAs across the different types of specimens could improve the early detection of NSCLC. METHODS: Using reverse transcription PCR, we determined expressions of two miRNAs (miRs-31-5p and 210-3p) in sputum and three miRNAs (miRs-21-5p, 210-3p, and 486-5p) in plasma of a training cohort of 76 NSCLC patients and 72 cancer-free smokers. The results were validated in a testing cohort of 56 NSCLC patients and 55 cancer-free smokers. RESULTS: The panels of two sputum miRNAs and three plasma miRNAs had 65.8-75.0% sensitivities and 83.3-87.5% specificities for diagnosis of NSCLC in the training cohort. The individual sputum or plasma miRNA panel had a higher sensitivity for squamous cell carcinoma or adenocarcinoma of the lung, respectively. From the miRNAs, we optimized an integrated panel of biomarkers consisting of two sputum miRNAs (miRs-31-5p and 210-3p) and one plasma miRNA (miR-21-5p) that had higher sensitivity (85.5%) and specificity (91.7%) for diagnosis of NSCLC compared with the individual panels alone. Furthermore, the performance of the integrated panel of biomarkers was independent of histology and stage of NSCLC, and patients' age, sex, and ethnicity. The performance of the integrated panel of biomarkers was confirmed in the testing cohort. CONCLUSIONS: Integrating biomarkers across different body fluids would synergistically improve the early detection of NSCLC. KEY POINTS: Lung cancer is a heterogeneous disease and develops from complex aberrations. Integrating sputum and plasma miRNAs has higher accuracy than when they are used alone.

Inhibition of cisplatin-resistant head and neck squamous cell carcinoma by combination of Afatinib with PD0325901, a MEK inhibitor.

ErbB family members that contain EGFR, HER2, HER3 and HER4 play important roles in many cancer types, including head and neck; however, inhibition of these receptors by small molecule kinase inhibitors showed limited results due to compensatory up-regulation of some key survival signaling pathways. Here, we explore the effectiveness of Afatinib, an irreversible inhibitor of EGFR, HER2, and HER4, in combination with the MEK inhibitor PD0325901 to inhibit cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We treated two cisplatin-resistant HNSCC cell lines, UMSCC74B and O28, with Afatinib, PD0325901, or a combination, and measured signaling pathways, cell proliferation, and survival. We found that Afatinib blocked Akt/mTOR activity and phosphorylation of EGFR, HER2 and HER3, but up-regulated MEK/ERK signaling. Interestingly, MEK inhibitor PD0325901 blocked ERK phosphorylation, but elevated phosphorylation of Akt and mTOR pathways. Similarly, Afatinib and PD0325901 inhibited all these pathways and synergistically suppressed cell proliferation and survival. Our data demonstrate that Afatinib in combination with MEK inhibitors could provide a potential novel therapy for cisplatin-resistant head and neck squamous cell cancer.

Regulation of cisplatin-resistant head and neck squamous cell carcinoma by the SRC/ETS-1 signaling pathway.

BACKGROUND: We investigated the role of the ETS-1 transcription factor in Head and Neck Squamous Cell Carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines. METHODS: We examined its molecular link with SRC and MEK/ERK pathways and determined the efficacy of either MEK/ERK inhibitor PD0325901 or SRC inhibitor Dasatinib on cisplatin-resistant HNSCC inhibition. RESULTS: We found that ETS-1 protein expression levels in a majority of cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin sensitive partners. High ETS-1 expression was also found in patient-derived, cisplatin-resistant HNSCC cells. While ETS-1 knockdown inhibited cell proliferation, migration, and invasion, it could still re-sensitize cells to cisplatin treatment. Interestingly, previous studies have shown that MER/ERK pathways could regulate ETS-1 through its phosphorylation at threonine 38 (T38). Although almost all cisplatin-resistant HNSCC cells we tested showed higher ETS-1 phosphorylation levels at T38, we found that inhibition of MEK/ERK pathways with the MEK inhibitor PD0325901 did not block this phosphorylation. In addition, treatment of cisplatin-resistant HNSCC cells with the MEK inhibitor completely blocked ERK phosphorylation but did not re-sensitize cells to cisplatin treatment. Furthermore, we found that, consistent with ETS-1 increase, SRC phosphorylation dramatically increased in cisplatin-resistant HNSCC, and treatment of cells with the SRC inhibitor, Dasatinib, blocked SRC phosphorylation and decreased ETS-1 expression. Importantly, we showed that Dasatinib, as a single agent, significantly suppressed cell proliferation, migration, and invasion, in addition to survival. CONCLUSIONS: Our results demonstrate that the SRC/ETS-1 pathway plays a crucial role and could be a key therapeutic target in cisplatin-resistant HNSCC treatment.

Co-targeting EGFR and IKKß/NF-κB signalling pathways in head and neck squamous cell carcinoma: a potential novel therapy for head and neck squamous cell cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) plays an important role in head and neck squamous cell carcinoma (HNSCC) proliferation and therapy resistance, but the efficacy of targeting of EGFR for therapy has been limited. Here, we explore the molecular link between EGFR and inhibitor of κB kinase ß/nuclear factor-κB (IKKß/NF-κB) signalling pathways in the regulation of HNSCC EGFR inhibitor resistance. METHODS: We performed in vitro experiments in eight human HNSCC cell lines and a patient-derived HNSCC cell line as well as in vivo xenografts in a human HNSCC cell line. RESULTS: We found that treatment of all HNSCC cells with Gefitinib and Erlotinib, two Food Drug Administration-approved EGFR inhibitors, blocked the activity of Akt/mammalian target of the rapamycin (mTOR) and extracellular signal-regulated kinase, two crucial downstream effectors of EGFR, but up-regulated IKKß/NF-κB signalling. In addition, induction of IKKß/NF-κB by EGFR inhibitors required HER2 and HER3 expression. In keeping with these, IKKß inhibitor CmpdA synergistically enhanced the efficacy of EGFR inhibitors to further inhibit in vitro HNSCC cell growth. Importantly, we demonstrated that the combination of Gefitinib with CmpdA inhibited xenograft tumour formation. CONCLUSION: Our data demonstrated that co-targeting EGFR and IKKß with Gefitinib and IKKß inhibitors could provide a potential novel therapy for head and neck squamous cell cancer.

Hepatitis C virus core impacts expression of miR122 and miR204 involved in carcinogenic progression via regulation of TGFBRAP1 and HOTTIP expression.

BACKGROUND: Despite the breadth of understanding the noncoding RNAs' function in molecular biology, their functional roles in hepatocellular carcinoma (HCC) is poorly understood. In this study, we investigated the effect of hepatitis C virus (HCV) core upon the expression of noncoding RNAs. METHODS: The lncRNAs, mRNAs, and circRNAs were employed for identification of HCV core protein gene expression in human Huh7 hepatoma (Huh7) cell line. In data analysis, we applied a threshold that eliminated all genes that were not increased or decreased by at least a 2-fold change in a comparison between transfected and control cells. Hierarchical Clustering and the Kyoto encyclopedia of genes and genome pathway analyses were performed to show the distinguishable lncRNA, mRNAs, and circRNAs expression pattern among samples. RESULTS: The array data showed that 4,851 lncRNAs, 4,785 mRNAs, and 823 circRNAs were 2-fold up-regulated but 3,569 lncRNAs, 3,192 mRNAs, and 419 circRNAs were 2-fold down-regulated in Huh 7-core cells. The genes in the enriched set were associated with macromolecule and nucleic acid metabolic processes, DNA damage response and regulation of voltage-gated calcium channel. We identified 10 genes from the selected 14 genes that were higher or lower expression in Huh7-core cells than that of Huh7-vector cells by quantitative real-time polymerase chain reaction. Interestingly, overexpression of miR122 and miR204 partly abrogated the expression of TGFBRAP1 and HOTTIP, and increased the HPCAL1 expression in the predicted carcinogenic pathways. CONCLUSION: Our data suggests that the pathways of miR204-HPCAL1-lncRNAHOTTIP and miR122-TGFBRAP1 were likely involved in the carcinogenic progress due to the presence of HCV core, and that overexpression of miR122 and miR204 might inhibit the HCC progress by down-regulation of TGFBRAP1 and HOTTIP expression.

Small nucleolar RNA signatures of lung tumor-initiating cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the number one cancer killer. Tumor-initiating cells (TICs) are responsible for tumor progression and recurrence. Emerging evidences suggest that small nucleolar RNAs (snoRNAs) play malfunctioning roles in lung tumorigenesis. This study aims to determine if snoRNAs have important function in lung TICs by: 1) profiling and comparing snoRNA expression patterns in lung ALDH1+/- cells of 28 primary NSCLC tissues to identify new signatures of TICs; 2) determining prognostic significance of the snoRNA signatures by analyzing the expression in 82 NSCLC tissues with different stages and histological types using quantitative PCR; 3) functionally investigating if the snoRNAs contribute to stemness of lung TICs using in vitro and in vivo assays. RESULTS: Twenty-two snoRNAs were identified whose changes were specific to the TICs. The expression of two snoRNAs (snoRA3 and snoRA42) was inversely associated with survival of NSCLC patients (P = 0.002, p = 0.001, respectively). Functional analysis indicated that snoRA42 was upregulated in CD133+ cells isolated from NSCLC cell lines compared with the CD133- counterparts. snoRA42 knockdown reduced the proliferation and self-renewal of TICs in vitro. However, ectopic expression of snoRA42 in non-TICs enhanced the potentials of cell proliferation and self-renewal. snoRA42 expression was associated with expression of stem cell-core transcription factors in lung TICs. Blocking snoRA42 expression in TIC xenografts decreased tumorigenesis in mice. CONCLUSIONS: The snoRNA signatures of lung TICs provide potential biomarkers for predicting outcome of NSCLC. snoRA42 is one of the important snoRNAs in regulating features of lung TICs, and thus contributes to lung tumorigenesis.

Analysis of MicroRNAs in sputum to improve computed tomography for lung cancer diagnosis.

INTRODUCTION: Computed tomography (CT) plays a central role in lung cancer diagnosis. However, CT has relatively low specificity, presenting a challenge in clinical settings. We previously identified 12 microRNAs (miRNAs) whose expressions in tumor tissues were associated with lung cancer. METHODS: Using quantitative reverse transcriptase polymerase chain reaction, we aimed to identify miRNA biomarkers in sputum that could complement CT for diagnosis of lung cancer. RESULTS: In a training set consisting of 66 lung cancer patients and 68 cancer-free smokers, 10 of the 12 miRNAs were differentially expressed between the cases and controls (p ≤ 0.01). From the miRNAs, a logistic regression model was built on the basis of miR-31 and miR-210, both of which had the best prediction for lung cancer, producing an area under receiver operating characteristic curve of 0.83. Combined use of the two miRNAs yielded 65.2% sensitivity and 89.7% specificity, CT had 93.9% sensitivity and 83.8% specificity for lung cancer diagnosis. Notably, combined analysis of the miRNA biomarkers and CT produced a higher specificity than does CT used alone (91.2% versus 83.8%; p < 0.05). The diagnostic performance of the biomarkers was confirmed in a testing set comprising 64 lung cancer patients and 73 cancer-free smokers. CONCLUSION: The sputum miRNA biomarkers might be useful in improving CT for diagnosis of lung cancer, but further independent validation on an external and prospective cohort of patients is required.

Small nucleolar RNAs in cancer.

Non-coding RNAs (ncRNAs) are important regulatory molecules involved in various physiological and cellular processes. Alterations of ncRNAs, particularly microRNAs, play crucial roles in tumorigenesis. Accumulating evidence indicates that small nucleolar RNAs (snoRNAs), another large class of small ncRNAs, are gaining prominence and more actively involved in carcinogenesis than previously thought. Some snoRNAs exhibit differential expression patterns in a variety of human cancers and demonstrate capability to affect cell transformation, tumorigenesis, and metastasis. We are beginning to comprehend the functional repercussions of snoRNAs in the development and progression of malignancy. In this review, we will describe current studies that have shed new light on the functions of snoRNAs in carcinogenesis and the potential applications for cancer diagnosis and therapy.

Diagnosis of lung cancer in individuals with solitary pulmonary nodules by plasma microRNA biomarkers.

BACKGROUND: Making a definitive preoperative diagnosis of solitary pulmonary nodules (SPNs) found by CT has been a clinical challenge. We previously demonstrated that microRNAs (miRNAs) could be used as biomarkers for lung cancer diagnosis. Here we investigate whether plasma microRNAs are useful in identifying lung cancer among individuals with CT-detected SPNs. METHODS: By using quantitative reverse transcriptase PCR analysis, we first determine plasma expressions of five miRNAs in a training set of 32 patients with malignant SPNs, 33 subjects with benign SPNs, and 29 healthy smokers to define a panel of miRNAs that has high diagnostic efficiency for lung cancer. We then validate the miRNA panel in a testing set of 76 patients with malignant SPNs and 80 patients with benign SPNs. RESULTS: In the training set, miR-21 and miR-210 display higher plasma expression levels, whereas miR-486-5p has lower expression level in patients with malignant SPNs, as compared to subjects with benign SPNs and healthy controls (all P ≤ 0.001). A logistic regression model with the best prediction was built on the basis of miR-21, miR-210, and miR-486-5p. The three miRNAs used in combination produced the area under receiver operating characteristic curve at 0.86 in distinguishing lung tumors from benign SPNs with 75.00% sensitivity and 84.95% specificity. Validation of the miRNA panel in the testing set confirms their diagnostic value that yields significant improvement over any single one. CONCLUSIONS: The plasma miRNAs provide potential circulating biomarkers for noninvasively diagnosing lung cancer among individuals with SPNs, and could be further evaluated in clinical trials.

Plasma microRNAs as potential biomarkers for non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Developing minimally invasive techniques that can diagnose NSCLC, particularly at an early stage, may improve its outcome. Using microarray platforms, we previously identified 12 microRNAs (miRNAs) the aberrant expressions of which in primary lung tumors are associated with early-stage NSCLC. Here, we extend our previous research by investigating whether the miRNAs could be used as potential plasma biomarkers for NSCLC. We initially validated expressions of the miRNAs in paired lung tumor tissues and plasma specimens from 28 stage I NSCLC patients by real-time quantitative reverse transcription PCR, and then evaluated diagnostic value of the plasma miRNAs in a cohort of 58 NSCLC patients and 29 healthy individuals. The altered miRNA expressions were reproducibly confirmed in the tumor tissues. The miRNAs were stably present and reliably measurable in plasma. Of the 12 miRNAs, five displayed significant concordance of the expression levels in plasma and the corresponding tumor tissues (all r>0.850, all P<0.05). A logistic regression model with the best prediction was defined on the basis of the four genes (miRNA-21, -126, -210, and 486-5p), yielding 86.22% sensitivity and 96.55% specificity in distinguishing NSCLC patients from the healthy controls. Furthermore, the panel of miRNAs produced 73.33% sensitivity and 96.55% specificity in identifying stage I NSCLC patients. In addition, the genes have higher sensitivity (91.67%) in diagnosis of lung adenocarcinomas compared with squamous cell carcinomas (82.35%) (P<0.05). Altered expressions of the miRNAs in plasma would provide potential blood-based biomarkers for NSCLC.

Small nucleolar RNA signatures as biomarkers for non-small-cell lung cancer.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC will improve its outcome. The current techniques for NSCLC early detection are either invasive or have low accuracy. Molecular analyses of clinical specimens present promising diagnostic approaches. Non-coding RNAs (ncRNAs) play an important role in tumorigenesis and could be developed as biomarkers for cancer. Here we aimed to develop small nucleolar RNAs (snoRNAs), a common class of ncRNAs, as biomarkers for NSCLC early detection. The study comprised three phases: (1) profiling snoRNA signatures in 22 NSCLC tissues and matched noncancerous lung tissues by GeneChip Array, (2) validating expressions of the signatures by RT-qPCR in the tissues, and (3) evaluating plasma expressions of the snoRNAs in 37 NSCLC patients, 26 patients with chronic obstructive pulmonary disease (COPD), and 22 healthy subjects. RESULTS: In the surgical tissues, six snoRNAs were identified, which were overexpressed in all tumour tissues compared with their normal counterparts. The overexpressions of the genes in tumors were confirmed by RT-qPCR. The snoRNAs were stably present and reliably detectable in plasma. Of the six genes, three (SNORD33, SNORD66 and SNORD76) displayed higher plasma expressions in NSCLC patients compared with the cancer-free individuals (All < 0.01). The use of the three genes produced 81.1% sensitivity and 95.8% specificity in distinguishing NSCLC patients from both normal and COPD subjects. The plasma snoRNA expressions were not associated with stages and histological types of NSCLC (All > 0.05). CONCLUSIONS: The identified snoRNAs provide potential markers for NSCLC early detection.

[Seasonal variation of fixed ammonium content of paddy soils in Hunan Province and its bioavailability].

Three paddy soils with higher content of fixed ammonium in Hunan Province were used as test soils in pot experiment to study the seasonal variation of their fixed ammonium content and its bioavailability during rice growth period. The results showed that the fixed ammonium content in the paddy soils changed constantly during rice growth. Applying N fertilizer and organic manure increased the fixed ammonium content, which was decreased through the N uptake by rice plants. The recently fixed ammonium was almost fully available to rice, while the native fixed ammonium was partly available. The release of fixed ammonium during rice growth was newly alluvial sandy soil > purple clayey soil > alluvial sandy soil, and was greater a early growth stage than at later growth stage.

[Fixed ammonium content of chief paddy soil types in Hunan Province and its influencing factors].

By the method of Silva and Bremner, the fixed ammonium content of chief paddy soil types in Hunan Province and its influencing factors were studied. The results showed that the content of fixed ammonium in plough layer ranged from 141 mg.kg-1 to 353 mg.kg-1, averaged 272 +/- 67 mg.kg-1, and accounted for 11.2% of total soil N. The content of fixed ammonium was in order of alluvial sandy soil > purple clayey soil > newly alluvial sandy soil > yellow clayey soil > reddish yellow clayey soil. There were four distribution models of fixed ammonium in the soil profiles: 1) fixed ammonium content increased with the increase of depth in the profiles; 2) fixed ammonium content decreased with the increase of depth; 3) there was no distinct change; and 4) abrupt increase or decrease was found in the profiles. The percentage of fixed ammonium to total N in soils always increased with the increase of depth. The fixation of NH4+ by soil was the strongest under 30 degrees C. Long-term submergence benefited to the fixation of NH4+ in newly alluvial sandy soil, purple clayey soil, and alluvial sandy soil, while the alternation of drying and wetting contributed to the fixation of NH4+ in yellow clayey soil best. The content of fixed ammonium in tested soil was correlated with the content of clay < 0.01 mm at the significant level, and not correlated with the content of clay < 0.001 mm, total N, organic N, and matter.