CLASRP oncogene as a novel target for colorectal cancer.

Clk4-associated serine/arginine-rich protein (CLASRP), an alternative splicing regulator, may be involved in the development and progression of cancer by regulating the activity of the CDC-like kinase (Clk) family. This study explored the biological function of CLASRP in colorectal cancer (CRC). The expression of CLASRP, which is associated with clinicopathological features, was analysed in CRC tissues and paired noncancer tissues by RT-PCR. The roles of CLASRP were investigated in CRC cells transfected with plasmids or shRNA through proliferation, migration and invasion assays in vitro and a xenograft model in vivo. Apoptosis was analysed using CLASRP-overexpressing CRC cells by western blotting. Clk inhibitors were used to perform functional research on CLASRP in CLASRP-overexpressing CRC cells. CLASRP was significantly upregulated in CRC cell lines, while high CLASRP expression was correlated with metastasis in CRC patients. Functionally, overexpression of CLASRP significantly promoted the proliferation, migration and invasion of CRC cells in vitro and tumour growth in vivo. Mechanistically, the proliferation, migration and invasion of CLASRP-overexpressing CRC cells were inhibited by Clk inhibitors, accompanied by low expression of CLASRP at the gene and protein levels. Clk inhibitors induced apoptosis of CLASRP-overexpressing CRC cells, resulting in direct blockade of cell growth. The expression levels of cleaved caspase 3 and cleaved caspase 8 were increased in CLASRP-overexpressing CRC cells treated with Clk inhibitors. CLASRP might serve as a promotional oncogene in CRC cells and be suppressed by Clk inhibitors through activation of caspase pathways.

Abnormal topological characteristics of brain white matter network relate to cognitive and emotional deficits of non-small cell lung cancer (NSCLC) patients prior to chemotherapy.

INTRODUCTION: Cancer and chemotherapy are correlated with brain functional and structural changes in cancer patients, which may lead to cognitive dysfunction. However, little is known about the structural abnormalities of brain in patients with non-small cell lung cancer (NSCLC). The aim of this study was to explore the topological properties within the brain white matter network of NSCLC patients prior to chemotherapy. METHODS: To explore the neurobiological biomarkers of NSCLC, brain magnetic resonance imaging (MRI) data were acquired in 24 non-nervous system metastatic NSCLC patients and 25 matched healthy controls. The topological properties of the brain structural networks of NSCLC were measured by the parameters of local and global efficiency. RESULTS: Treatment-naïve NSCLC patients showed cognitive and emotional deficits. In addition, NSCLC patients also exhibited decreased global efficacy in the left inferior frontal gyrus (triangular part), left inferior frontal gyrus (orbital part), right rolandic operculum, right gyrus rectus, right lenticular nucleus (putamen), left superior temporal gyrus and right inferior temporal gyrus. Decreased local efficacy were found in the left middle frontal gyrus (orbital part) and left superior temporal gyrus in NSCLC patients. Moreover, the aberrant brain regions were associated with the impaired cognitiion and emotion of NSCLC patients. CONCLUSION: Overall our results suggested that altered local and global efficiency of brain white matter network were associated with cancer-induced cognitive and emotional deficits of NSCLC patients. These findings demonstrated that disrupted topological characteristics of the brain network might underlie the impaired cognition and emotion in NSCLC.

New genetic variations discovered in KRAS wild-type cetuximab resistant chinese colorectal cancer patients.

To perform a comprehensive genomic analysis of colorectal cancer (CRC) tumor to detect genetic variants and identify novel resistant mutations associated with cetuximab-resistance in CRC patients. A retrospective study was performed using whole exome sequencing (WES) to identify common genetic factors from 22 cetuximab-sensitive and 10 cetuximab-resistant patients. In all 10 cetuximab-resistant patients, we discovered there are 37 significantly mutated genes (SMGs). CYP4A11 was the most frequently mutated gene in cetuximab-resistant patients. BCAS1 and GOLGA6L1 were found to be among the second group of frequently mutated genes with a frequency of 60%. After cosine similarity analysis, three mutational signatures (signature a, b, and c) were found in all CRC tumors, similar to signature 1, 5, and 6 in COSMIC, respectively. Gene ontology analysis was performed on SMGs and found 12 enriched GO terms. Four genes are enriched in six specific Kyoto Encyclopedia of Genes and Genomes pathway groups, including the metabolism of xenobiotics by cytochrome P450, steroid hormone biosynthesis, retinol metabolism, and drug metabolism. Our data supports a network composed of SMGs and cellular signaling pathways that have been positively linked to the mechanisms of cetuximab resistance. These involve DNA damage repair, angiogenesis, invasion, drug metabolism, and the CRC tumor microenvironment. There is a SMG, OR9G1 correlated with survival rates of KRAS wild-type colon adenocarcinoma patients. These findings support further investigation using WES in a prospective clinical study of cetuximab resistance CRC, to further identify, confirm, and extend the clinical significance of these and other potentially important new candidate predictive biomarkers of cetuximab response.

Comparison of droplet digital PCR and direct Sanger sequencing for the detection of the BRAFV600E mutation in papillary thyroid carcinoma.

BACKGROUND: The BRAFV600E mutation status is a useful diagnostic and prognostic marker for papillary thyroid carcinoma (PTC). Although it is a commonly used method, Sanger sequencing has several limitations in detecting the BRAFV600E mutation. The aim of this study was to evaluate the efficiency of droplet digital PCR (ddPCR) as an alternative method for the detection of the BRAFV600E mutation in PTC patients. METHODS: Samples from a total of 120 patients with PTC and 30 patients with benign nodular thyroid disease who underwent thyroid surgery were collected. The BRAFV600E mutation status of the PTC patients was tested by Sanger sequencing and ddPCR. RESULTS: The BRAFV600E mutation was detected in 67 samples (44.67%) by Sanger sequencing and 92 samples (61.33%) by ddPCR. The detection of the mutation by the two methods was inconsistent in twenty-five samples (16.67%). The sensitivity and specificity of the ddPCR method were 100% and 69.88%, respectively, and the positive predictive and negative predictive values were 72.83% and 100%, respectively. The concordance rate between the two methods in detecting the BRAFV600E mutation was 83.33%. Neither Sanger sequencing nor ddPCR detected BRAFV600E in 30 patients with benign nodular thyroid disease. The 92 samples with the BRAFV600E mutation were detected by ddPCR at a fractional abundance from 0.28% to 45.40% as follows: ≥10% (59 samples, 64.13%), 5%-10% (8 samples, 8.70%), and ≤5% (25 samples, 27.17%). The BRAFV600E mutation was detected in all 59 samples at a fractional abundance ≥10% and in four samples at a fractional abundance from 5% to 10%, and no BRAFV600E mutation was detected at a fractional abundance ≤5% by Sanger sequencing. CONCLUSIONS: ddPCR was a reliable, highly sensitive alternative method for the detection of the BRAFV600E mutation in PTC patients.

Hepcidin in tumor-related iron deficiency anemia and tumor-related anemia of chronic disease: pathogenic mechanisms and diagnosis.

OBJECTIVES: Anemia is a common hematological abnormality in patients with cancer. Iron deficiency anemia (IDA) and anemia of chronic disease (ACD) are the most prevalent, both characterized by hypochromic microcytic anemia and low serum iron (SI). Their differential diagnosis is difficult in clinical practice, hampering their treatment. Our objective was to evaluate the use of hepcidin to discriminate tumor-related IDA and ACD and to investigate the mechanism of action of hepcidin in these anemias. METHODS: Blood samples were collected at Jiangsu Cancer Hospital. Patients were divided into IDA and ACD groups by Prussian blue staining of bone marrow smears. Serum hepcidin was measured by enzyme-linked immunosorbent assay. SI, total iron-binding capacity (TIBC), transferrin saturation (TSAT), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) also determined in this study. RESULTS: Areas under the curve on receiver operating characteristic analysis indicated the diagnostic sensitivity and specificity of hepcidin to be better than those of SI, TIBC, and TSAT. In ACD, hepcidin was correlated positively with IL-6 (r = 0.81, P < 0.01) and negatively with SI (r = -0.78, P < 0.01). In IDA, no significant relationship between IL-6 and hepcidin was found (r = -0.20, P = 0.17), but hepcidin decreased with decreasing quartiles of SI (r = 0.89, P < 0.01). SI was positively correlated with hemoglobin (r = 0.89, P < 0.01; r = 0.84, P < 0.01) in both groups. CONCLUSIONS: Hepcidin is a promising serological marker for the differential diagnosis of tumor-related ACD and IDA, clarifying the pathogenesis of these anemias and guiding corrective treatment.

The function role of miR-181a in chemosensitivity to adriamycin by targeting Bcl-2 in low-invasive breast cancer cells.

OBJECTIVES: miR-181a is involved in immunity, metabolism, tumor suppression or carcinogenesis reported by many other studies. However, its role in the development of chemosensitivity to adriamycin in low-invasive breast cancer cells remains unclear. The aim of this study is to define the function role of miR-181a in promoting the efficacy of adriamycin-based neoadjuvant chemotherapy. METHODS: Cell survival analysis was detected by Cell Counting Kit-8 assay. Apoptotic cells were quantitatively detected using FITC Annexin V apoptosis Detection Kit I. Bcl-2 protein expression was measured by western blot. Luciferase reporter vector with the putative BCL-2 3' untranslated region (3'UTR) was constructed to explore whether BCL-2 was a direct target gene of miR-181a. Real-time PCR was performed to test the expression of miR-181a and Bcl-2 in the selected breast cancer tissue samples. RESULTS: The down-regulation of miR-181a decreased adriamycin-induced apoptosis in MCF-7 cells. Transfected with miR-181a mimic in cells resulted in the decreased expression of Bcl-2. The alteration of miR-181a expression did not significantly affect the chemosensitivity to adriamycin in MCF-7 and MCF-7/ADR cells with genetic knockout of Bcl-2. miR-181a may suppress Bcl-2 expression by forming imperfect base pairing with the 3'UTR of Bcl-2 gene such that a negative relationship between miR-181a and Bcl-2 in MCF-7 and MCF-7/ADR cells is observed. CONCLUSIONS: At least in part, the detection of miR-181a may direct the clinical medication in patients with neoadjuvant chemotherapy because of miR-181a enhanced adriamycin-induced apoptosis via targeting Bcl-2.

Serum VEGF levels as a predictor of recurrence in patients with advanced­stage esophageal squamous cell carcinoma following curative esophagectomy followed by chemotherapy or concurrent radiotherapy.

The present study evaluated serum levels of vascular endothelial growth factor (VEGF) as a predictor of recurrence in patients with advanced-stage esophageal squamous cell carcinoma (ESCC) following curative esophagectomy followed by chemotherapy or concurrent radiotherapy. Patients with locally advanced resectable ESCC underwent R0 esophagectomy followed by chemotherapy or concurrent radiotherapy as an adjuvant. Serum VEGF levels in 173 patients, including 57 patients with recurrent disease, and 183 healthy controls were determined using a Luminex assay. The results demonstrated that the serum VEGF levels were significantly higher in 57 patients with locally advanced resectable ESCC at recurrence compared with the levels at pre-treatment (P<0.001). The patients with recurrence exhibited significantly higher serum VEGF levels during chemotherapy or concurrent radiotherapy than patients with no recurrence (P<0.05). Patients with low serum VEGF levels had a significantly longer survival time than those with high serum VEGF levels prior to treatment (P<0.01). The median survival times were 70 and 25 months in patients with locally advanced resectable ESCC with serum VEGF levels <161.75 and ≥161.75 pg/ml following treatment, respectively (P<0.01). Compared with patients with VEGF levels <147 pg/ml following treatment, patients with locally advanced resectable ESCC with VEGF levels ≥147 pg/ml had a significantly higher risk of recurrence (P<0.01). Patients with low serum VEGF levels (<147 pg/ml) had significantly higher recurrence-free survival rates than those with high serum VEGF levels (≥147 pg/ml) following treatment (P<0.01). The findings of the present study demonstrate that serum VEGF levels are a potential predictor of recurrence and of the treatment outcomes of chemotherapy or concurrent radiotherapy in patients with locally advanced resectable ESCC.

Nedaplatin reduces multidrug resistance of non-small cell lung cancer by downregulating the expression of long non-coding RNA MVIH.

Cisplatin-based chemotherapy is the standard treatment for non-small cell lung cancer (NSCLC). However, drug resistance emergences after treatment. Long non-coding RNA microvascular invasion in hepatic cancer (MVIH) plays an important role in drug resistance in a variety of cancers. This study investigates the role of nedaplatin on multidrug resistance in NSCLC and its relationship with MVIH. Lung cancer A549 and H1650 cells were treated with cisplatin to obtain multidrug-resistant A549/DDP and H1650/ DDP cells. A549/DDP and H1650/ DDP cells were treated with nedaplatin, MVIH siRNA and siRNA NC. It was found that both MVIH siRNA and nedaplatin significantly reduce the mRNA expression of MVIH in A549/DDP and H1650/ DDP cells. MTT assay showed that the proliferation of MDR cells was significantly higher than that of other cells. Nedaplatin and MVIH siRNA significantly inhibit the proliferation of A549 and H1650 cells. The results of colony formation assay were consistence with MTT results. Nedaplatin and MVIH siRNA significantly reduced colony formation in MDR cells. Flow cytometry showed that NDP and MVIH siRNA significantly decrease the proportion of cells in G0/G1 and increase the proportion of cells in S phase compared with untreated and MDR cells. The apoptotic rate of MDR cells was significantly lower than that of other cells, while the apoptosis rate of cells in NDP and MVIH siRNA group was significantly higher than that of the other three groups of cells. Wound healing assay and Transwell chamber experiments confirmed that both NDP and MVIH siRNA significantly reduced the migration and invasion abilities of MDR cells. The expression of E-cadherin in MDR cells was significantly lower than that in untreated cells, and the expression of N-cad, α-SMA and Vimentin significantly increased in the MDR cells. NPD and MVIH siRNA reverse the EMT process. In conclusion, LncRNA MVIH is upregulated in drug resistant NSCLC cells. Nedaplatin can reduce the expression of MVIH and reverse EMT process, thus reversing the drug resistance of cisplatin in non-small cell lung cancer cells.

Cancer-associated changes of emotional brain network in non-nervous system metastatic non-small cell lung cancer patients: a structural connectomic diffusion tensor imaging study.

BACKGROUND: Emotional distress frequently occur in cancer patients following diagnosis. Previous neuroimaging studies have demonstrated that depression and anxiety are associated with functional and structural brain abnormalities. However, little is known about the cancer-associated changes of emotional brain network in non-small cell lung cancer (NSCLC) patients. The aim of this study was to assess the topological features of brain structural network and emotions in non-nervous system metastatic NSCLC patients prior to chemotherapy. METHODS: Twenty-four treatment-naïve patients with non-nervous system metastatic NSCLC and 25 healthy controls (HC) matched for gender, age and education participated in this study. All subjects underwent diffusion tensor imaging (DTI), and were assessed with the 17 item hamilton depression rating scale (HAMD-17) and hamilton anxiety rating scale (HAMA). Properties of brain network were examined by the method of graph-theoretic analysis. The assessments included small-worldness, clustering coefficient and shortest path length. RESULTS: NSCLC patients had higher scores of HAMD-17 and HAMA when compared with HC. Additionally, we found a small-world topology of brain white matter network in both NSCLC and HC. NSCLC patients had significantly reduced clustering coefficient compared to healthy controls in the left hippocampus. Moreover, increased shortest path length were identified in NSCLC patients, which included the left middle frontal gyrus (orbital part), superior temporal gyrus and right Rolandic operculum, rectus gyrus, lenticular nucleus (putamen). However, no correlations were found between the impaired brain regions and HAMD-17, HAMA scores of NSCLC patients. CONCLUSIONS: Our results indicated impaired topological characteristics in the brain structural network of non-nervous system metastatic NSCLC patients prior to chemotherapy, which might account for the cancer-related emotional distress. Our findings demonstrated that NSCLC might affect brain regions involved in the process of emotion, which identified the basis of emotional changes associated with cancer.

The somatic mutation landscape of Chinese Colorectal Cancer.

Colorectal cancer (CRC) is the fifth leading cause of cancer-related death in China. The incidence of Chinese CRC has increased dramatically with the changes of dietary and lifestyle. However, the genetic landscape of Chinese colorectal cancer mutation is still poorly understood. In this study, we have performed whole exome-sequencing analysis of 63 CRC cases. We found that Chinese CRC were hypermutated, which were enriched in ECM-receptor interaction, antigen processing and presentation, and focal adhesion. Analysis with clinical characteristics indicated that the deficiency of CRC driver gene, FCGBP and NBPF1 conferred CRC development and was showed worse survival rates, which could be the novel regulators and, diagnostic and prognostic biomarkers for Chinese CRC. Taken together, the application of whole exome-sequencing unveiled previously unsuspected somatic mutation landscape in Chinese CRCs, which may expand the understanding of disease mechanisms and provide an alternative personalized treatment for Chinese CRC patients.

Monoclonal antibody AC10364 inhibits cell proliferation in 5-fluorouracil resistant hepatocellular carcinoma via apoptotic pathways.

BACKGROUND: This study was designed to investigate the antitumor activity of the mAb (AC10364) in vitro and elucidate the related mechanisms of inhibition to cell growth using bel/fu cells treated with AC10364. METHODS: The inhibitory effects of AC10364 on the proliferation of Bel/fu cells were examined using a cytotoxicity assay. Apoptosis of Bel/fu cells was detected using FITC annexin V and PI staining following treatment with AC10364 for 24 h. The factors regulating apoptosis were identified by Western blot using lysates of Bel/fu cells treated with AC10364 for 0, 12, 24, or 36 h. Genes associated with tumorigenesis or growth were analyzed by reverse transcription-quantitative polymerase chain reaction using Bel/fu cells treated for 12, 24, or 36 h with AC10364. RESULTS: The early apoptotic ratios of Bel/fu cells treated with AC10364 increased in a dose-dependent manner. The levels of caspases, including cleaved caspase-3, caspase-3 and caspase-9, were significantly high in Bel/fu cells treated with AC10364 (P<0.001). Compared with untreated cells, those exposed to AC10364 had showed significant downregulation of the expression of binding protein gene (G protein subunit α 15, GNA15) and other protein-coding genes, including fms-related tyrosine kinase 1(FLT1), nicotinamide phosphoribosyltransferase (NAMPT), netrin 4 (NTN4), platelet-derived growth factor subunit A (PDGFA), S100 calcium binding protein A11 (S100A11), tubulin ß 3 class III (TUBB3), aldo-keto reductase family 1 member C3 (AKR1C3), endothelial PAS domain protein 1 (EPAS1), and interferon α-inducible protein 27 (IFI27) (P<0.001). Two other genes, AXL receptor tyrosine kinase (AXL) and carboxypeptidase A4 (CPA4), were significantly upregulated (P<0.001). CONCLUSION: AC10364 inhibited cell viability and proliferation through aberrant expression of multiple genes associated with tumorigenesis or growth, which suggests that these genes may be promising therapeutic candidates for cancer therapy.

Long noncoding RNA and mRNA profiling in cetuximab-resistant colorectal cancer cells by RNA sequencing analysis.

To gain an insight into the molecular mechanisms of cetuximab resistance in colorectal cancer, we generated a cetuximab-resistant cell line (H508/CR) and performed RNA sequencing to identify the differential expression patterns of noncoding RNAs (ncRNAs) and mRNAs between cetuximab-sensitive and resistant cells. A total of 278 ncRNA transcripts and 1,059 mRNA transcripts were dysregulated in the cetuximab-resistant cells. The expression levels of nine selected long noncoding RNAs (lncRNAs) were validated using quantitative real-time PCR. Functional analysis revealed that several groups of lncRNAs might be involved in pathways associated with cetuximab resistance. Increased glucose consumption and lactate secretion in cetuximab-resistant cells suggested that glucose metabolism might be involved in cetuximab resistance. In addition, lncRNA LINC00973 was upregulated in the H508/CR cell line and cells transfected with a LINC00973 short interfering RNA exhibited reduced cell viability, increased apoptosis, and decreased glucose consumption and lactate secretion. Our results provide essential data regarding differentially expressed lncRNAs and mRNAs in cetuximab-resistant cells, which may provide new potential candidates for cetuximab therapy.

Circulating tumor DNA detection: A potential tool for colorectal cancer management.

Colorectal cancer (CRC) is frequently diagnosed at an advanced stage of the disease, the pathogenesis of which is influenced by genetic and epigenetic events. Circulating tumor DNA (ctDNA) is extracellular DNA that is present in a number of bodily fluids, including blood, synovial fluid and cerebrospinal fluid. Compared with performing a tissue biopsy, ctDNA examination presents the advantages of minimal invasion and greater convenience. ctDNA is commonly used to identify actionable genomic alterations, monitor treatment responses, unravel therapeutic resistance and potentially detect disease progression prior to clinical and radiological confirmation. The technique can potentially serve as a non-invasive diagnostic tool in personalized medicine, as it demonstrates prognostic value in the management of patients with CRC. ctDNA detection continues to demonstrate inherent advantages compared with other methods, thus serving an increasingly important role in tumor monitoring and oncotherapy. The aim of the current review was to explore the clinical applications of ctDNA in patients with CRC, including early detection and screening, medication guidance, resistance prediction, and residual lesion and recurrence monitoring. Furthermore, several technical methods for ctDNA detection and analysis are explored, as well as other potential biomarkers.

MEK inhibitor enhanced the antitumor effect of oxaliplatin and 5­fluorouracil in MEK1 Q56P­mutant colorectal cancer cells.

Mitogen­activated protein kinase kinase (MEK) small molecule inhibitors have been investigated in preclinical or clinical trials for the treatment of cancer. In the present study the genetic test results of 120 patients with colorectal cancer (CRC) were screened and the mutation rate of MEK1 was identified to be 1.67%. MEK inhibition by U0126 significantly decreased the growth of SW48 cells that harbored the MEK1 Q56P mutation, although it did not evidently affect the growth of NCI­H508 cells with MEK1 wild­type. In addition, U0126 increased the sensitivity of SW48 cells to 5­fluorouracil (5­FU) and oxaliplatin by producing more γH2AX foci and decreasing the expression of excision repair cross­complementation group 1 and thymidylate synthase. The results suggested that MEK inhibitors in combination with oxaliplatin/5­FU may offer an improved therapeutic effect in patients with MEK­mutant CRC.

Next­generation sequencing­based detection of EGFR, KRAS, BRAF, NRAS, PIK3CA, Her­2 and TP53 mutations in patients with non­small cell lung cancer.

In recent years, the incidence of non­small cell lung cancer (NSCLC) has become the highest lethal rate of cancer worldwide. Molecular assays of EGFR, KRAS, BRAF, NRAS, PIK3CA and Her­2 are widely used to guide individualized treatment in NSCLC patients. Somatic mutations in 112 NSCLC patients, including 7 oncogenic driver genes, were detected by Iontorrent personal genome machine (PGM). Sanger sequencing was used to test and verify the results of PGM. Apart from uncommon mutations of EGFR, 101 NSCLC specimens were tested by droplet digital PCR (ddPCR). According to NGS results, mutations were detected in EGFR (58/112, 51.79% of tumors), KRAS (10/112, 8.93%), BRAF (2/112, 1.79%), NRAS (2/112, 1.79%), Her­2 (2/112, 1.79%), PIK3CA (6/112, 5.36%) and TP53 (31/112, 27.69%). There were 27 samples without any somatic mutations in all genes while 24 samples harboured mutations in two or more genes. A total of 61 samples had one or more mutations in a single gene. All alterations of 7 genes were presented and the overall detection rate of NGS and Sanger sequencing was determined to be 51.79% (58/112) and 37.50% (42/112), respectively (χ2=5.88, P=0.015). Compared with Sanger sequencing, the total sensitivity and specificity of NGS assays was 95.24% (40/42) and 77.14% (54/70), respectively. The overall detection rate of NGS and ddPCR was 45.54% (46/101) and 47.52% (48/101), respectively (χ2=0.000598, P=0.98). Compared with ddPCR, the overall sensitivity and specificity of NGS assays was 95.83% (46/48) and 98.11% (52/53), respectively. The findings indicated that the positive mutation rate of EGFR tested by NGS was significantly lower than that by Sanger sequencing, but the difference between ddPCR and NGS was not statistically significant. The high degree of agreement of reportable variants is proposed in both NGS and ddPCR analysis, suggesting the performance of NGS assays in routine clinical detection may be useful in determining the treatment decisions in NSCLC patients.

Exosome-mediated gefitinib resistance in lung cancer HCC827 cells via delivery of miR-21.

Acquired resistance to gefitinib remains a major challenge in cancer treatment. In the present study, the effect of exosomes on the transmission of gefitinib resistance from gefitinib-resistant HCC827 lung cancer cells (H827R) to their gefitinib-sensitive counterparts and the potential underlying mechanisms by which this occurs was investigated. Exosomes were obtained from the cell supernatant using ultracentrifugation and the ExoQuick-TC exosome precipitation solution. Drug resistance was assessed by flow cytometry, apoptosis assays and cell counting kit-8 assays. The expression of microRNA (miR)-21 was analyzed by reverse transcription-quantitative polymerase chain reaction. Exosomes released by H827R cells (R/exo) may decrease the sensitivity of the human NSCLC HCC827 cell line to gefitinib. The results indicated that miR-21 expression was increased in R/exo and R/exo-treated H827S cells. However, miR-21 inhibition abrogated exosome-mediated drug resistance. Phosphorylated-protein kinase B (p-Akt), which is downstream of miR-21, was downregulated following gefitinib treatment; however, R/exo pretreatment elevated p-Akt levels and promoted the activation of Akt. By contrast, miR-21 inhibition reduced p-Akt expression. Therefore, the induction of miR-21 via exosomes and the activation of Akt may be mechanisms by which exosomes mediate the transfer of drug resistance.

LXR ligands induce apoptosis of EGFR-TKI-resistant human lung cancer cells in vitro by inhibiting Akt-NF-κB activation.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are efficient in treating patients with non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. Unfortunately, nearly all patients ultimately acquire resistance to EGFR-TKI treatment. Liver X receptors (LXRs) can regulate tumor growth in various cancer cell lines. The present study indicated that LXR agonist combined with gefitinib weakened Akt-nuclear factor (NF)-κB activation and inhibited the expression levels of apoptosis-related proteins in vitro. By contrast, LXR ligands alone exhibited no significant effect on gefitinib-resistant lung cells. In conclusion, the study provided evidence for the combination treatment of acquired TKI resistance in NSCLC.

Cytokine-induced killer cells as a feasible adoptive immunotherapy for the treatment of lung cancer.

Most of the patients with lung cancer are diagnosed at advanced stage, and they often lose the opportunity of surgical therapy, most of whom fail to reach good prognosis after chemotherapy. Recently, a few clinical studies have confirmed the role of adoptive T-cell transfer in the maintenance therapy of cancer patients. Here, we provided statistical insights into the role of CIKs in advanced lung cancer from three different levels, cell model (in vitro co-culture system), mice model (in situ lung cancer), and clinical research (in lung cancer patients of different progression stages). We optimized the components of supplements and cytokines on activating and expanding CIK cells. Based on this, we explored a new serum-free medium for in vitro activation and expansion of CIK cells. Moreover, we found that activated CIK cells could efficiently kill lung cancer cells in cell-to-cell model in vitro and significantly reduce the tumor growth in mice. For the clinical research, the OS rates of patients received combination of chemotherapy and CIK treatment were significantly improved compared to the OS rates of patients only received chemotherapy. Additionally, CIK therapy represented good toleration in our study. All the results suggested that combination of immunotherapy with traditional therapy will be a feasible and promising method for the treatment of lung cancer.

Liver X receptor agonist T0901317 reverses resistance of A549 human lung cancer cells to EGFR-TKI treatment.

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is effective in lung cancer patients carrying sensitive EGFR mutations. In this study, we investigated if liver X receptor (LXR) agonist T0901317 could reverse the resistance of lung cancer cell lines A549 and H1650 to EGFR-TKI treatment. We found that T0901317 could make natural EGFR-TKI-resistant A549 human lung cancer cells sensitive to EGFR-TKI treatment and that this was dependent on LXRß expression. However, T0901317 does not have a similar effect on another natural EGFR-TKI-resistant cell line H1650.

Identification of serum proteins and multivariate models for diagnosis and therapeutic monitoring of lung cancer.

Lung cancer is one of the most prevalent cancers and has very poor treatment outcome. Biomarkers useful for screening and assessing early therapeutic response may significantly improve the therapeutic outcome but are still lacking. In this study, serum samples from 218 non-small cell lung cancer (NSCLC) patients, 34 small cell lung cancer (SCLC) patients and 171 matched healthy controls from China were analyzed for 11 proteins using the Luminex multiplex assay. Eight of the 11 proteins (OPN, SAA, CRP, CYFRA21.1, CEA, NSE, AGP and HGF) are significantly elevated in NSCLC and SCLC (p = 10-5-10-59). At the individual protein level, OPN has the best diagnostic value for NSCLC (AUC = 0.92), two acute phase proteins (SAA and CRP) have AUC near 0.83, while CEA and CYFRA21.1 also possess good AUC (0.81 and 0.77, respectively). More importantly, several three-protein combinations that contain OPN and CEA plus one of four proteins (CRP, SAA, CYFRA21.1 or NSE) have excellent diagnostic potential for NSCLC (AUC = 0.96). Four proteins (CYFRA21.1, CRP, SAA and NSE) are severely reduced and three proteins (OPN, MIF and NSE) are moderately decreased after platinum-based chemotherapy. Therapeutic response index (TRI) computed with 3-5 proteins suggests that approximately 25% of the NSCLC patients respond well to the therapy and TRI is significantly correlated with pre-treatment protein levels. Our data suggest that therapeutic response in NSCLC patients can be effectively measured but personalized biomarkers may be needed to monitor different subsets of patients.