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1.  Copy number changes of 4-gene set may predict early relapse in advanced epithelial ovarian cancer after initial platinum-paclitaxel chemotherapy 
For advanced epithelial ovarian cancer (EOC), time to recurrence (TTR) is an important indicator to gauge the therapeutic efficacy of postoperative adjuvant chemotherapy. Our objective was to determine the genes that could potentially distinguish patients with short versus long TTR after initial administration of platinum-paclitaxel combination chemotherapy in advanced EOC. Tumor samples of 159 patients were obtained during the primary cytoreduction. Array comparative genomic hybridization (CGH) was carried with genomic DNA from 17 EOC samples (8 with TTR > 15 months and 9 with TTR ≤ 6 months) to screen candidate gene set, copy-number changes (CNC) of which were significantly different between early and late relapse cases. Seventeen candidate genes were identified by array CGH. The analysis of consistency between real-time PCR and array CGH revealed that 4 genes displayed consistent results, namely GSTT1, ISG20L1, STARD5 and FREM1. In a 142-case validation set, CNC of 4 candidate genes was evaluated and verified by real-time PCR. Sixty five point five percent of the patients were correctly divided into early (TTR ≤ 10 months) and late (TTR > 10 months) recurrent group by CNC of the 4 genes using discriminant analysis. The results showed that CNC of 4-gene set could potentially determine early (TTR ≤ 10 months) or late relapse (TTR > 10 months) after initial platinum-paclitaxel combination chemotherapy in advanced EOC.
PMCID: PMC4065409  PMID: 24959383
Epithelial ovarian cancer; paclitaxel; platinum; recurrence; array comparative genomic hybridization
2.  Imbalanced expression of Tif1γ inhibits pancreatic ductal epithelial cell growth 
Transcriptional intermediary factor 1 gamma (Tif1γ) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGFβ pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1γ interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1γ inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1γ and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1γ resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1γ resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1γ reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1γ results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1γ may inhibit tumor invasion. These data suggest that Tif1γ might serve as a potential therapeutic target for pancreatic cancer.
PMCID: PMC4065401  PMID: 24959375
Transcriptional intermediary factor 1 gamma (Tif1γ); Smad; pancreatic cancer
3.  Histone/protein deacetylase SIRT1 is an anticancer therapeutic target 
SIRT1, a member of the NAD+-dependent histone/protein deacetylase family, is involved in chromatin remodeling, DNA repair, and stress response and is a potential drug target. 5-fluorouracil (FU) and the SN1-type DNA methylating agent temozolomide (TMZ) are anticancer agents. In this study, we demonstrate that sirt1 knockout mouse embryonic fibroblast cells are more sensitive to FU and DNA methylating agents than normal cells. Based on these findings, the chemotherapy efficacy of SIRT1 inhibitors in combination with FU or TMZ were tested with human breast cancer cells. We found that treatments combining SIRT1 inhibitors with FU or TMZ show synergistic reduction of cell viability and colony formation of breast cancer cells. Thus, inhibition of SIRT1 activity provides a novel anticancer strategy.
PMCID: PMC4065402  PMID: 24959376
Breast cancer; drug resistance; SIRT1 histone deacetylase; 5-fluorouracil; methylating agents
4.  A novel recombinant papillomavirus genome enabling in vivo RNA interference reveals that YB-1, which interacts with the viral regulatory protein E2, is required for CRPV-induced tumor formation in vivo  
YB-1 is considered a negative prognostic marker for different types of cancer. Increased YB-1 protein levels in tumor cells indicate a worse prognosis. In a preceding study comparing the transcripts of CRPV-induced benign papillomas to mRNA levels of malignant epithelial tumors, we identified YB-1 as a gene that is up-regulated in papillomavirus-associated carcinomas and which causes an invasive phenotype in CRPV-positive cells in vitro. Here we demonstrate that YB-1 is a previously unknown factor required for papillomavirus-induced tumor development in the rabbit animal model system. By infecting the animals with a novel recombinant shRNA-expressing CRPV genome, we show that knock-down of YB-1 dramatically reduces papillomavirus-dependent tumor formation in vivo. Consistent with previous reports showing a nuclear distribution of YB-1 proteins as a hallmark of malignancy, we demonstrate a predominantly nuclear localization of YB-1 in CRPV-immortalized cells. Furthermore we give evidence of YB-1 regulating the CRPV URR and thereby viral gene expression and we identified YB-1 as a novel interactor of the CRPV regulatory protein E2. Taken together we hypothesize that YB-1 is essential for papillomavirus-induced tumor formation probably by regulating viral gene expression including expression of the oncogenes E6 and E7.
PMCID: PMC4065403  PMID: 24959377
Papillomavirus; CRPV; rabbit; in vivo; YB-1; E2; viral transcription; protein-protein interaction
5.  Research findings working with the p53 and Rb1 targeted osteosarcoma mouse model 
Osteosarcoma (OS) is the most common bone cancer in children and young adults. The etiology of osteosarcoma is currently unknown. Besides the predominant osteoblasts, the presence of cartilage forming chondrocytes within its tumor tissues suggests a role of chondrogenesis in osteosarcoma development. Runx2 is a master transcription factor both for osteoblast differentiation and for chondrocyte maturation. Interestingly, RUNX2 has been shown to directly interact with p53 and Rb1, two genes essential for osteosarcoma development in mice. However the in vivo relevance of Runx2 during osteosarcoma progression has not been elucidated. We have recently shown that targeting Runx2 expression in hypertrophic chondrocytes delays chondrocyte maturation. It has also been shown that osteoblast-specific deletion of p53 and Rb1 genes developed osteosarcoma in mice. Here, we report our recent research findings using these osteosarcoma mouse models as well as human osteosarcoma tissues. We have detected high-level RUNX2 expression in human osteoblastic osteosarcoma, while chondroblastic osteosarcoma is predominant with chondroid matrix. To minimize the effect of strain difference, we have backcrossed osterix-Cre mice onto congenic FVB/N genetic background. We also detected low-GC content (36%) in sequence around the floxed Rb1 gene and demonstrated that addition of BSA into the reaction system increases the efficiency of PCR genotyping of floxed Rb1 gene. Finally, we successfully generated multiple osteosarcoma mouse models with or without Runx2 transgenic background. These mice showed heterogeneous osteosarcoma phenotypes and marker gene expression. Characterization of these mice will facilitate understanding the role of Runx2 in osteosarcoma pathogenesis and possibly, for osteosarcoma treatment.
PMCID: PMC4065404  PMID: 24959378
Osteosarcoma mouse model; p53 and Rb1; Runx2; BSA; PCR genotyping
6.  SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation 
The deacetylase SIRT1 regulates multiple biological processes including cellular metabolism and aging. Importantly, SIRT1 can also inactivate the p53 tumor suppressor via deacetylation, suggesting a role in oncogenesis. Recently, SIRT1 was shown to be released from its endogenous inhibitor DBC1 by a process requiring AMPK and the phosphorylation of SIRT1 by yet undefined kinase(s). Here we provide further evidence that AMPK directly phosphorylates SIRT1 on T344, releasing it from DBC1. Furthermore, a phospho-mimetic SIRT1 (T334E) showed decreased binding to DBC1, supporting the importance of this phosphorylation in AMPK-mediated regulation of SIRT1 activity. In addition, inhibition of AMPK by Compound C led to increased p53 acetylation, suggesting a role for the AMPK/SIRT1 pathway in regulating p53 signaling. Together, our results support a hypothesis that AMPK negatively regulates p53 acetylation via phosphorylation of SIRT1 on T344. Furthermore, our findings also define the AMPK/SIRT1 axis as a possible targetable pathway to regulate p53 function.
PMCID: PMC4065405  PMID: 24959379
AMPK; SIRT1 deacetylase; DBC1; p53; phosphorylation; acetylation
7.  Protein signature for non-small cell lung cancer prognosis 
Background: Current histopathological classification and TNM staging have limited accuracy in predicting survival and stratifying patients for appropriate treatment. The goal of the study is to determine whether the expression pattern of functionally important regulatory proteins can add additional values for more accurate classification and prognostication of non-small lung cancer (NSCLC). Methods: The expression of 108 proteins and phosphoproteins in 30 paired NSCLC samples were assessed using Protein Pathway Array (PPA). The differentially expressed proteins were further confirmed using a tissue microarray (TMA) containing 94 NSCLC samples and were correlated with clinical data and survival. Results: Twelve of 108 proteins (p-CREB(Ser133), p-ERK1/2(Thr202/Tyr204), Cyclin B1, p-PDK1(Ser241), CDK4, CDK2, HSP90, CDC2p34, β-catenin, EGFR, XIAP and PCNA) were selected to build the predictor to classify normal and tumor samples with 97% accuracy. Five proteins (CDC2p34, HSP90, XIAP, CDK4 and CREB) were confirmed to be differentially expressed between NSCLC (n=94) and benign lung tumor (n=19). Over-expression of CDK4 and HSP90 in tumors correlated with a favorable overall survival in all NSCLC patients and the over-expression of p-CREB(Ser133) and CREB in NSCLC correlated with a favorable survival in smokers and those with squamous cell carcinoma, respectively. Finally, the four proteins (CDK4, HSP90, p-CREB and CREB) were used to calculate the risk score of each individual patient with NSCLC to predict survival. Conclusion: In summary, our data demonstrated a broad disturbance of functionally important regulatory proteins in NSCLC and some of these can be selected as clinically useful biomarkers for diagnosis, classification and prognosis.
PMCID: PMC4065406  PMID: 24959380
Lung cancer; biology marker; survival analysis
8.  Expression of migfilin is increased in esophageal cancer and represses the Akt-β-catenin activation 
Cell adhesion proteins that connect each cell to neighboring cells and the extracellular matrix are an important determinant of cell morphology and metastasis. Migfilin is a member of LIM-containing protein family, mediated the linkage between cytoskeleton and focal adhesion through interacting with other proteins and involved in cell adhesion and motility. The present study used immunohistochemistry and Western blot analysis of migfilin in clinical specimens of esophageal squamous cell carcinoma (ESCC) to identify that the expression of migfilin was significantly upregulated in ESCC in concomitance with a nuclear-cytoplasm translocation compared to normal adjacent tissue. Alternately, using the published datasets we identified that the expression of β-catenin was upregulated in esophageal cancer cells with focal invasion, while inversely correlated with migfilin in esophageal cancer cell lines. We demonstrated that the reduced expression of β-catenin by migfilin was through the inhibition of Akt activation. In conclusion, these results illustrated that migfilin upregulated in ESCCs and repressed β-catenin in an Akt-GSK3β signaling dependent manner.
PMCID: PMC4065407  PMID: 24959381
Migfilin; β-catenin; Akt; GSK3β; cell adhesion; esophageal cancer
9.  Impairment of aldehyde dehydrogenase 2 increases accumulation of acetaldehyde-derived DNA damage in the esophagus after ethanol ingestion 
Ethanol and its metabolite, acetaldehyde, are the definite carcinogens for esophageal squamous cell carcinoma (ESCC), and reduced catalytic activity of aldehyde dehydrogenase 2 (ALDH2), which detoxifies acetaldehyde, increases the risk for ESCC. However, it remains unknown whether the ALDH2 genotype influences the level of acetaldehyde-derived DNA damage in the esophagus after ethanol ingestion. In the present study, we administered ethanol orally or intraperitoneally to Aldh2-knockout and control mice, and we quantified the level of acetaldehyde-derived DNA damage, especially N2-ethylidene-2’-deoxyguanosine (N2-ethylidene-dG), in the esophagus. In the model of oral ethanol administration, the esophageal N2-ethylidene-dG level was significantly higher in Aldh2-knockout mice compared with control mice. Similarly, in the model of intraperitoneal ethanol administration, in which the esophagus is not exposed directly to the alcohol solution, the esophageal N2-ethylidene-dG level was also elevated in Aldh2-knockout mice. This result indicates that circulating ethanol-derived acetaldehyde causes esophageal DNA damage, and that the extent of damage is influenced by knockout of Aldh2. Taken together, our findings strongly suggest the importance of acetaldehyde-derived DNA damage which is induced in the esophagus of individuals with ALDH2 gene impairment. This provides a physiological basis for understanding alcohol-related esophageal carcinogenesis.
PMCID: PMC4065408  PMID: 24959382
Carcinogenesis; esophageal squamous cell carcinoma; acetaldehyde; acetaldehyde-derived DNA damage; DNA adduct
10.  Genome-wide copy number analysis in pediatric glioblastoma multiforme 
Glioblastoma (GBM) is a very aggressive and lethal brain tumor with poor prognosis. Despite new treatment strategies, patients’ median survival is still less than 1 year in most cases. Few studies have focused exclusively on this disease in children and most of our understanding of the disease process and its clinical outcome has come from studies on malignant gliomas in childhood, combining children with the diagnosis of GBM with other pediatric patients harboring high grade malignant tumors other than GBM. In this study we investigated, using array-CGH platforms, children (median age of 9 years) affected by GBM (WHO-grade IV). We identified recurrent Copy Number Alterations demonstrating that different chromosome regions are involved, in various combinations. These observations suggest a condition of strong genomic instability. Since cancer is an acquired disease and inherited factors play a significant role, we compared for the first time the constitutional Copy Number Variations with the Copy Number Alterations found in tumor biopsy. We speculate that genes included in the recurrent 9p21.3 and 16p13.3 deletions and 1q32.1-q44 duplication play a crucial role for tumorigenesis and/or progression. In particular we suggest that the A2BP1 gene (16p13.3) is one possible culprit of the disease. Given the rarity of the disease, the poor quality and quantity of bioptic material and the scarcity of data in the literature, our findings may better elucidate the genomic background of these tumors. The recognition of candidate genes underlying this disease could then improve treatment strategies for this devastating tumor.
PMCID: PMC4065410  PMID: 24959384
Pediatric glioblastoma multiforme; central nervous tumor; copy number alterations (CNA); copy number variations (CNVs); array-CGH; minimum common regions; deletion; duplication; amplification; tumorigenesis
11.  Anticancer drug FL118 is more than a survivin inhibitor: where is the Achilles’ heel of cancer? 
Can a solution be found that overcomes all chemotherapy and/or radiation resistance resulting from different genetic and epigenetic alternations in various cancer types? The answer is likely NO. However, there are two ways that may be followed to approach this goal. One way is through the use of poly-therapies that target multiple mechanisms to kill cancer cells, which is the current state of the art. This approach raises issues of high costs and/or toxic limitations, since the toxicities of each agent are often additive. This poly-pharmacy approach has not proven to be a major success, although it has proven to be superior to most current mono-pharmacy approaches. The other way to approach the goal is to find a single anticancer drug that targets multiple different treatment resistant mechanisms. In this regard, a small chemical molecule (FL118) was recently discovered by serendipity during targeted discovery of anticancer drugs using the survivin gene as a target and biomarker. FL118 was found to not only inhibit multiple antiapoptotic proteins (survivin, XIAP, cIAP2) in the inhibitor of apoptosis (IAP) family, but to also inhibit the antiapoptotic protein Mcl-1 in the Bcl-2 family, while inducing the pro-apoptotic proteins Bax and Bim expression. Importantly, inhibition of these target genes and of tumor growth by FL118 is independent of p53 status (wild type, mutant or null), although mechanisms of action may be distinct among cells with different p53 status. Therefore, FL118 may effectively control cancer that loses functional p53, in which most DNA damage drugs (if not all) show a marked lack of efficiency. Recent studies further revealed that the superior anticancer activity of FL118 is highly dependent on its primary structure and steric configuration, suggesting that FL118 may be a promising drug platform for generating novel derivatives based on its core structure. Intriguingly, although FL118 has structural similarity to irinotecan and topotecan, two FDA-approved topoisomerase 1 (Top1) inhibitors for cancer treatment, cancer cells with Top1 mutations shows little contributions of treatment resistance to FL118 antitumor activity, while strikingly increasing irinotecan and topotecan resistance. Furthermore, both irinotecan and topotecan are the efflux pump ABCG2 substrates; cancer cells with high expression of ABCG2 showed strong irinotecan and topotecan resistance. In contrast, FL118 is not an ABCG2 substrate; ABCG2 overexpression in cancer cells does not show resistance to FL118 treatment. Current evidence suggests that future studies may unravel more unexpected mechanisms of action for this unique small molecule FL118.
PMCID: PMC4065411  PMID: 24959385
Anticancer; drug; FL118; survivin inhibitor; the inhibitor of apoptosis; topoisomerase 1
12.  S100 protein family in human cancer 
S100 protein family has been implicated in multiple stages of tumorigenesis and progression. Among the S100 genes, 22 are clustered at chromosome locus 1q21, a region frequently rearranged in cancers. S100 protein possesses a wide range of intracellular and extracellular functions such as regulation of calcium homeostasis, cell proliferation, apoptosis, cell invasion and motility, cytoskeleton interactions, protein phosphorylation, regulation of transcriptional factors, autoimmunity, chemotaxis, inflammation and pluripotency. Many lines of evidence suggest that altered expression of S100 proteins was associated with tumor progression and prognosis. Therefore, S100 proteins might also represent potential tumor biomarkers and therapeutic targets. In this review, we summarize the evidence connecting S100 protein family and cancer and discuss the mechanisms by which S100 exerts its diverse functions.
PMCID: PMC3960449  PMID: 24660101
S100 proteins; proliferation; apoptosis; invasion; migration; pluripotency; biomarker
13.  Polyisoprenylated methylated protein methyl esterase overexpression and hyperactivity promotes lung cancer progression 
The involvement of hyperactive polyisoprenylated proteins in cancers has stimulated the search for drugs to target and suppress their excessive activities. Polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition has been shown to modulate polyisoprenylated protein function. For PMPMEase inhibition to be effective against cancers, polyisoprenylated proteins, the signaling pathways they mediate and/or PMPMEase must be overexpressed, hyperactive and be involved in at least some cases of cancer. PMPMEase activity in lung cancer cells and its expression in lung cancer cells and cancer tissues were investigated. PMPMEase was found to be overexpressed and significantly more active in lung cancer A549 and H460 cells than in normal lung fibroblasts. In a tissue microarray study, PMPMEase immunoreactivity was found to be significantly higher in lung cancer tissues compared to the normal controls (p < 0.0001). The mean scores ± SEM were 118.8 ± 7.7 (normal), 232.1 ± 25.1 (small-cell lung carcinomas), 352.1 ± 9.4 (squamous cell carcinomas), 311.7 ± 9.8 (adenocarcinomas), 350.0 ± 24.2 (papillary adenocarcinomas), 334.7 ± 30.1 (adenosquamous carcinomas), 321.9 ± 39.7 (bronchioloalveolar carcinomas), and 331.3 ± 85.0 (large-cell carcinomas). Treatment of lung cancer cells with L-28, a specific PMPMEase inhibitor, resulted in concentration-dependent cell death (EC50 of 8.5 μM for A549 and 2.8 μM for H460 cells). PMPMEase inhibition disrupted actin filament assembly, significantly inhibited cell migration and altered the transcription of cancer-related genes. These results indicate that elevated PMPMEase activity spur cell growth and migration, implying the possible use of PMPMEase as a protein biomarker and drug target for lung cancer.
PMCID: PMC3960450  PMID: 24660102
Polyisoprenylation; esterase; Ras; lung cancer; isoprenylation; methylation; nanostring; monomeric G-proteins
14.  Sepantronium is a DNA damaging agent that synergizes with PLK1 inhibitor volasertib 
In a search for novel agents that boost the anti-neoplastic effects of polo-like kinase 1 (PLK1) inhibitor volasertib, we found that a sepantronium and volasertib combination at the nano mole concentration potently inhibited growth of various non-small cell lung cancer (NSCLC) cell lines than either drug alone in vitro. Combination use of volasertib with sepantronium inhibited adaptation of cells to polo arrest. Addition of sepantronium to volasertib prevented accumulation of survivin and cyclin B protein at a concentration causing no appreciable survivin down regulation. Sepantronium induced cell cycle arrest from G1 or G2/M phase. Further studies demonstrated DNA damage of cancer cells when they are treated with sepantronium, which is evidenced by induction of phospho-γH2AX. In line with induction of a DNA damage response in cancer cells, known DNA damage response sensors and transducers ATM, ATR, CHK1, CHK2, p53 are phosphorylated following drug treatment. Meanwhile, expression of CDKN1A, BAX and XRCC5 are induced at the mRNA level as determined by quantitative real time PCR. A single cell electrophoresis assay (Comet assay) of cells treated with sepantronium revealed severe DNA strand breaks. M-phase arrest does not increase the lethality of DNA damage by sepantronium as compared to G1 phase arrest. Knock down of survivin did not cause DNA damage. Hence, sepantronium is a DNA damaging agent that synergizes with volasertib and down-regulation of survivin is likely the consequence of DNA damage induced by sepantronium.
PMCID: PMC3960451  PMID: 24660103
Sepantronium; volasertib; DNA damage; lung cancer; synergy
15.  UMMS-4 enhanced sensitivity of chemotherapeutic agents to ABCB1-overexpressing cells via inhibiting function of ABCB1 transporter 
Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters through efflux of antineoplastic agents from cancer cells is a major obstacle to successful cancer chemotherapy. The inhibition of these ABC transporters is thus a logical approach to circumvent MDR. There has been intensive research effort to design and develop novel inhibitors for the ABC transporters to achieve this goal. In the present study, we evaluated the ability of UMMS-4 to modulate P-glycoprotein (P-gp/ABCB1)-, breast cancer resistance protein (BCRP/ABCG2)- and multidrug resistance protein (MRP1/ABCC1)-mediated MDR in cancer cells. Our findings showed that UMMS-4, at non-cytotoxic concentrations, apparently circumvents resistance to ABCB1 substrate anticancer drugs in ABCB1-overexpressing cells. When used at a concentration of 20 μmol/L, UMMS-4 produced a 17.53-fold reversal of MDR, but showed no effect on the sensitivity of drug-sensitive parental cells. UMMS-4, however, did not significantly alter the sensitivity of non-ABCB1 substrates in all cells and was unable to reverse ABCG2- and ABCC1-mediated MDR. Additionally, UMMS-4 profoundly inhibited the transport of rhodamine 123 (Rho 123) and doxorubicin (Dox) by the ABCB1 transporter. Furthermore, UMMS-4 did not alter the expression of ABCB1 at the mRNA and protein levels. In addition, the results of ATPase assays showed that UMMS-4 stimulated the ATPase activity of ABCB1. Taken together, we conclude that UMMS-4 antagonizes ABCB1-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1. These findings may be useful for the development of safer and more effective MDR modulator.
PMCID: PMC3960452  PMID: 24660104
UMMS-4; multidrug resistance; ATP binding cassette transporters; ABCB1; chemotherapeutic drugs
16.  Assessment of M867, a selective caspase-3 inhibitor, in an orthotopic mouse model for non-small cell lung carcinoma 
Radiation-induced lung injury (RILI) is a significant dose limiting complication of thoracic radiation for lung, breast, and esophageal cancer. Strategies for increasing the therapeutic index of radiation involve the use of radiosensitizing agents. We investigated the potential of M867 to sensitize non-small cell lung cancer (NSCLC) to radiation in vivo, while assessing its protective effects in normal lung parenchyma. H460-Luc2 cells were implanted into the mediastinum of athymic nude mice, which were separated into four treatment groups: control, M867, radiation therapy (RT) or combination. H460-Luc2 cell cultures were treated in parallel. Tumor growth was followed using bioluminescence imaging. Immunohistochemistry staining was used to detect phospho-Smad2/3 and cleaved caspase-3 expression. Western blot was done for the detection of cleaved caspase-3 and phospho-Smad2/3. TUNEL assays were used to measure apoptosis. M867+RT group had significantly increased tumor growth inhibition relative to either treatment alone (p=0.02). M867+RT was associated with increased levels of apoptosis (p<0.01), but combination treatment was associated with a decrease in caspase-dependent apoptosis relative to RT alone (p<0.01). We found that this increase in apoptosis in the M867+RT group was due to caspase-independent cell death. Based on early biomarker analyses of phospho-Smad 2/3 and cleaved caspase-3, M867+RT had a radio-protective effect on normal lung parenchyma. M867 may increase the therapeutic ratio of RT by enhancing the radiosensitivity of NSCLC while mitigating RILI. Further research is warranted to examine the late effects of lung injury and to study differences in the mechanism of action of M867 on lung cancer and normal tissue.
PMCID: PMC3960453  PMID: 24660105
M867; NSCLC; orthotopic mouse model; caspase; H460-Luc2; radiation
17.  Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model 
Combination therapies for melanoma that target immune-regulatory networks are entering clinical practice, and more are under investigation in preclinical or clinical studies. Adenosine plays a key role in regulating melanoma progression. We investigated the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody (mAb) in combination with either modulators of adenosine receptors (AR) activation or an inhibitor of adenosine production in a murine model of melanoma. We found that treatment with APCP, selective inhibitor of the adenosine-generating nucleotidase CD73, enhanced the activity of anti-CTLA4 mAb, by improving tumor immune response. Blockade of the adenosine A2a receptor (A2aR), which plays a critical role in the regulation of T-cell functions, significantly reduced melanoma growth. Most importantly, combination therapy including an A2aR antagonist with anti-CTLA4 mAb markedly inhibited tumor growth and enhanced anti-tumor immune responses. Targeting A3R and CTLA4 was not as effective in limiting melanoma growth as targeting A2aR. These data suggest that the efficacy of anti-CTLA4 melanoma therapy may be improved by targeting multiple mechanisms of immune suppression within tumor tissue, including CD73 or A2a receptor.
PMCID: PMC3960454  PMID: 24660106
CD73; adenosine receptor; CTLA4; melanoma; immunotherapy
18.  GNG2 inhibits invasion of human malignant melanoma cells with decreased FAK activity 
It is well known that heterotrimeric G protein is composed of a Gα-subunit and a Gβγ-dimer and promotes cancer characteristics. Our recent study showed reduced G protein γ2 subunit (Gng2/GNG2) expression levels in malignant melanoma cells compared with those in benign melanocytic cells in both mice and humans. Our recent study also showed that reduced GNG2 alone augmented proliferation of malignant melanoma cells. To our knowledge, however, there is no evidence showing an effect of Gng2/GNG2 alone on metastasis of any cancers including malignant melanoma. In his study, we first prepared GNG2-overexpressed SK-Mel28 human malignant melanoma cells, in which GNG2 protein expression level was undetectably low. Migration and invasion activities of the GNG2-overexpressed malignant melanoma cells were suppressed up to 1/10th, with decreased activity of focal adhesion kinase (FAK). We then found that the expression level of GNG2 in A375M, a highly metastatic cell line, was significantly lower than that in A375P, the parental cell line of A375M. We finally showed that knockdown of GNG2 alone in A375P cells enhanced migration and invasion with increased FAK activity. Taken together, our results suggest that overexpression of GNG2 alone inhibits metastasis in human malignant melanoma cells with decreased FAK activity. Thus, GNG2 might be a candidate of molecular targets of prevention and therapy for metastasis of malignant melanoma.
PMCID: PMC3960455  PMID: 24660107
G-protein; gamma subunit; malignant melanoma; invasion
19.  Significance of peripheral neutrophil-lymphocyte ratio among gastric cancer patients and construction of a treatment-predictive model: a study based on 1131 cases 
Gastric cancer (GC) is one of the most common and deadly malignancies nowadays, and inflammatory cells are closely related to tumor progression. This prospective study aims to uncover clinical significance of peripheral immune cells and build a treatment-predictive model. From July 2006 to July 2011, a total of 1131 GC patients were selected, with their general characteristics, peripheral blood and pathological parameters, and operational information obtained. The relevancies between preoperational neutrophil-lymphocyte ratio (NLR) and postsurgical pathological indexes were analyzed. SPSS 17.0 was applied in data analysis, comparing the differences of NLR between different groups using Mann-Whitney U test, contrasting the pathological differences between NLR elevated and reduced groups using Fisher test, and quantifying the correlation between post-surgical pathology and pre-operational NLR using univariate analysis. Patients were then classified into radical (applied in the training dataset) and non-radical gastrectomy (applied in the test dataset) groups, based on which we further tried to build a predictive model indicating appropriateness for radical resection using support vector machine (SVM). We found that: patients with tumor invading out of the myometrium (pT3-4) had significantly larger NLR than those with lesion limited within the myometrium (pT1-2) (P<0.05); poorly differentiated and undifferentiated malignancies were associated with higher NLR than well and moderately differentiated ones (P<0.05); there was larger NLR among patients with tumor length ≥4 cm than those <4 cm (P<0.01); preoperative NLR was significantly positively correlated with tumor TNM classification, number of metastatic lymph nodes, invasive depth and tumor size (P<0.05); larger proportion of elevated NLR was significantly associated with larger tumor size, later tumor and nodal stages, and higher TNM classification (P<0.01). We finally built a SVM model based on peripheral carcinoembryonic antigen, carbohydrate antigen 19-9, lymphocyte percentage and platelet count, effectively predicting the inappropriateness of patients undergoing curative gastrectomy when all the 4 parameters elevated with high accuracy (74.61% for the training dataset and 75.28% for the test dataset). We concluded that peripheral blood NLR indicated tumor progression, and that an efficient treatment-predictive SVM model was constructed.
PMCID: PMC3960456  PMID: 24660108
Gastric carcinoma; neutrophil-lymphocyte ratio; support vector machine; gastrectomy; tumor progression
20.  Beyond laser microdissection technology: follow the yellow brick road for cancer research 
Normal biological tissues harbour different populations of cells with intricate spacial distribution patterns resulting in heterogeneity of their overall cellular composition. Laser microdissection involving direct viewing and expertise by a pathologist, enables access to defined cell populations or specific region on any type of tissue sample, thus selecting near-pure populations of targeted cells. It opens the way for molecular methods directed towards well-defined populations, and provides also a powerful tool in studies focused on a limited number of cells. Laser microdissection has wide applications in oncology (diagnosis and research), cellular and molecular biology, biochemistry and forensics for tissue selection, but other areas have been gradually opened up to these new methodological approaches, such as cell cultures and cytogenetics. In clinical oncology trials, molecular profiling of microdissected samples can yield global “omics” information which, together, with the morphological analysis of cells, can provide the basis for diagnosis, prognosis and patient-tailored treatments. This remarkable technology has brought new insights in the understanding of DNA, RNA, and the biological functions and regulation of proteins to identify molecular disease signatures. We review herein the different applications of laser microdissection in a variety of fields, and we particularly focus attention on the pre-analytical steps that are crucial to successfully perform molecular-level investigations.
PMCID: PMC3902229  PMID: 24482735
Laser microdissection; histopathology; quality control; snap-freezing; DNA; RNA; proteomics; in situ cellular and molecular analyses
21.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
22.  Mutation of the retinoblastoma tumor suppressor gene sensitizes cancers to mitotic inhibitor induced cell death 
The retinoblastoma gene Rb is a prototype tumor suppressor, which encodes a protein that is inactivated in a broad range of human cancers through different mechanisms. Rb functions to regulate cell proliferation, differentiation, as well as cell death. Therefore, even though Rb inactivation promotes cancer development, this may also open up certain vulnerabilities of cancers that can potentially be targeted with drug intervention. Based on the assumption that cancers that have mutation, deletion, or rearrangement in the Rb locus represent strong loss of Rb function while cancers with WT Rb on average retain some Rb function, we searched Genomics of Drug Sensitivity in Cancer database to identify cancer drugs that are particularly effective to cancers with Rb genomic alterations. Three mitotic inhibitors were identified from this analysis. We further tested the effects of two mitotic inhibitors, Taxol and STLC, on prostate and breast cancer cells. We demonstrate that the Rb status affects cancer cell sensitivity to these mitotic drugs and that the sensitizing effects of Rb are mediated in part by its regulation of the cell cycle checkpoint protein Mad2. Since the mitotic inhibitors identified in our analysis inhibit mitosis through distinct targets, it is possible that the Rb functional status may serve as a general biomarker for cancer sensitivity to mitotic inhibitors. Because the Rb pathway is inactivated in a large number of human cancers, identification of agents that are particularly effective or ineffective based on the Rb status in cancers can potentially be used generally to matching patients with appropriate treatments to achieve better therapeutic outcome.
PMCID: PMC3902231  PMID: 24482737
Drug sensitivity; Rb; retinoblastoma tumor suppressor; Mad2; cell death; mitotic inhibitor; Taxol; S-Trityl-L-cysteine; STLC
23.  A novel experimental hypoxia chamber for cell culture 
Tissue hypoxia is a common pathophysiological process. Since 1990s, numerous studies have focused on investigating cellular adaptation to experimental hypoxia. A modular incubator chamber made of solid materials has frequently been used in the experiments that require hypoxic conditions. Here, we introduce a novel and inflatable chamber for hypoxia experiments. In experiments detecting hypoxia-induced accumulation of hypoxia-inducible factor 1α (HIF-1α) and hypoxia-induced expression of HIF-1-regulated genes, the new chamber yielded reproducible and comparable results as the modular incubator chamber did. The new chamber did not create inner chamber pressure during its use. Other properties of the new chamber were low-cost, easy to use, and leakage-free. Moreover, the size of the new chamber was adjustable, and the smaller one could be placed onto an inverted microscope for real-time studies. The successful examples of real-time studies included the real-time recording of GFP-HIF-1α fusion nuclear translocation and endothelial cell tubular formation.
PMCID: PMC3902232  PMID: 24482738
Cell culture; hypoxia; hypoxia chamber; hypoxia-inducible factor 1
24.  Biomarkers and endosalpingiosis in the ovarian and tubal microenvironment of women at high-risk for pelvic serous carcinoma 
Introduction: BRCA mutations increase the risk for development of high-grade pelvic serous carcinomas. Tissue biomarkers distinguishing women at high-risk (HR) for ovarian cancer from those at low-risk (LR) may provide insights into tumor initiation pathways. Methods: A prospective study of 47 HR women (40% BRCA carriers) undergoing risk-reducing salpingo-oophorectomy and 48 LR controls undergoing salpingo-oophorectomy was performed. Ovarian/tubal tissues were harvested. Immunohistochemical analysis of candidate proteins CSF-1, CSF-1R, ErbB4 is presented, with scores separately analyzed in epithelium and stroma, in ampulla, fimbria, ovary, and ovarian endosalpingiosis (ES). Comparison was performed between HR and LR groups. Results: Elevated levels of CSF-1 (p=0.005) or ErbB4 (p=0.005) in the ovarian epithelium, or ErbB4 (p=0.005) in the ovarian stroma, were significantly associated with both the HR status and carrying a BRCA mutation, as was nuclear ErbB4 staining. Ovarian ES, an entity which likely derives from the tubal mucosal epithelium, was also associated with HR (p=0.038) and BRCA mutation status (p=0.011). Among the BRCA carriers only, markers also found association when present in the tube as well as in ovarian ES (p < 0.05). ROCs were generated including in the regression model both CSF-1 and ErbB4 expression levels. A model including CSF-1 in ovarian epithelium, ErbB4 in ovarian stroma, and younger age achieves AUC=0.87 (73% sensitivity, 93% specificity) of detection of the HR status. In BRCA carriers, CSF-1 in ovarian epithelium alone achieves AUC=0.85. Conclusions: Our data suggest that elevated levels of CSF-1/ErbB4 in the adnexae correlate with HR/BRCA carrier status. CSF-1/CSF-1R signaling is active in ovarian cancer progression; our data suggests a role in its initiation. ErbB4, in particular nuclear ErbB4, may have a role in tumor initiation as well. Ovarian ES, an entity which may represent a latent precursor to low-grade pelvic serous carcinomas, was surprisingly associated with both HR status and the BRCA carrier cohort. In line with these findings, both ErbB4 and CSF-1R expression in ovarian ES correlated with carrying a BRCA mutation. This analysis, which needs to be validated, indirectly suggests a potential link between ovarian ES and the development of pelvic serous carcinoma in women who are BRCA mutation carriers.
PMCID: PMC3902233  PMID: 24482739
CSF-1; ErbB4; endosalpingiosis; high-risk
25.  PCOS and obesity: insulin resistance might be a common etiology for the development of type I endometrial carcinoma 
Endometrial cancer (EC) is the most common gynecological malignancy in women and is the leading cause of cancer-related deaths worldwide. Estrogenic stimulation significantly increases endometrial cell proliferation, and both insulin resistance and hyperinsulinemia are associated with the development of EC in women. It has long been known that insulin resistance occurs in women with polycystic ovary syndrome (PCOS) and/or obesity, but one important unanswered question is whether the insulin resistance associated with PCOS and obesity is part of the etiology of the initiation and development of EC. Therefore, research efforts to understand the common and specific underlying endometrial responses to insulin resistance in women with PCOS and obesity could provide further therapeutic options for early endometrial carcinoma.
PMCID: PMC3902234  PMID: 24482740
PCOS; obesity; insulin resistance; estrogen; IGF-1; endometrial carcinoma

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