Adjacent Normal-appearing Tissue Research Articles

Long Noncoding RNAs-LET Behave as a Noncoding Signature for Early-Onset Menarche and Late-Onset Menopause in Breast Cancer Patients

Background: Breast cancer (BC) as a major cause of cancer-related death in women shows a very complex molecular and clinical phenotype, which has reduced the effectiveness of medical interventions. Evidence suggests that long noncoding RNAs (lncRNAs) are responsible for an important part of this complexity. This study aims to assess the expression and clinical implication of lncRNA LET in the pathobiology of BC. Materials and Methods: Quantitative real-time polymerase chain reaction was used to measure the expression of lncRNA-LET in breast tumors and adjacent normal-appearing tissues from 4 BC patients, as well as normal mammary tissues. Moreover, a bioinformatics approach was applied to uncover the potential lncRNA-LET-mediated sponge regulatory network as LET/miRNA/mRNA crosstalk. Results: Our study revealed that lncRNA-LET was significantly down-expressed not only in breast tumors but also in normal appearing breast tissues samples from BC subjects compared with true normal breast tissues obtained from healthy women. The low level of lncRNA-LET was meaningfully associated with early-onset menarche (≤13 years) and late-onset menopause (≥50) in patients. Moreover, the bioinformatics analyses support that lncRNA-LET could function as a tumor suppressor miRNA sponge. Conclusion: The results indicate that normal appearing breast tissues can undergo tumor-related molecular changes. Furthermore, they reveal the potential role of the dysregulation in LET-mediated ceRNA network in the pathophysiology of BC.

Accumulation of plasmacytoid dendritic cell is associated with a treatment response to DNA-damaging treatment and favorable prognosis in lung adenocarcinoma.

Favorable responses to the treatment including immune checkpoint inhibitors (ICIs) have been consistently reported in lung cancer with smoking history. As the tumor microenvironment (TME) may be involved in the treatment response to ICIs, we aimed to investigate the TME of lung cancer with different smoking status. Lung adenocarcinoma (LUAD) tissue (Tu) and adjacent normal-appearing lung tissue (NL) from current and never smokers were investigated by single-cell RNA sequencing and immunofluorescence and immunohistochemical staining. The clinical implications of identified biomarkers were validated using open-source datasets. The lungs of smokers had an increased proportion of innate immune cells in NL tissues, whereas Tu tissues had a lower proportion of these cells than those of non-smokers. Monocyte-derived macrophages (mono-Mc), CD163-LGMN macrophages, monocyte-derived dendritic cells (DCs), and plasmacytoid DCs (pDCs) were significantly enriched in smokers' Tu. Among these clusters, pDCs, specifically enriched in the Tu of smokers. The expression of representative pDC markers, leukocyte immunoglobulin-like receptor A4 (LILRA4) and Toll-like receptor 9 (TLR9), was increased in the stromal cells of LUAD in patients with a smoking history. In an animal model of lung cancer, ionizing radiation induced robust TLR9 expressing immune cells in peritumoral area. Survival analysis using a TCGA-LUAD dataset indicated that patients overexpressing pDC markers exhibited superior clinical outcomes to age-, sex-, and smoking-matched control groups. Top 25% patients with high TLR9 expression exhibited significantly higher tumor mutational burden than that of low TLR9 expression group (bottom 25% patients) (5.81 mutations/Mb vs 4.36 mutations/Mb; P = 0.0059, Welch's two-sample t-test). There is an increased pDC in the TME of smokers' lung cancer, and the response of pDC to DNA damaging treatment would lead a conducive environment to ICIs containing regimens. These findings suggest that R&D that induces an increase in the activated pDC population is continuously required to enhance therapeutic effectiveness of ICIs-containing therapies in lung cancer.

The Generation and Application of Monoclonal Antibodies to Detect SAPCD2 Expression in Precancerous and Malignant Gastric Lesions.

Suppressor APC domain containing 2 (SAPCD2) is involved in cell cycle regulation and its mRNA levels are higher in cancer tissues. But, the function of SAPCD2 in cancer development remains unclear. To generate mouse monoclonal antibodies (mAbs) specific to SAPCD2 and thus clarify the function of SAPCD2 in the development of gastric carcinoma (GC). Methods: Purified SAPCD2-GST immunized BALB/c mouse spleen cells were collected and fused with myeloma cells to obtain monoclonal antibody hybridoma. A group of monoclonal antibodies exhibiting high specificity and sensitivity against SAPCD2 has been generated and characterized by IHC, WB, IP, IF, and ELISA. By immunohistochemical analysis, the SAPCD2 expression was evaluated in 228 clinical samples of gastric mucosal lesions, including precancerous lesions and GC samples. We identified a highly specific and sensitive clone of s12 in eukaryotic cells and performed an IHC analysis. We found that 81.3% of 107 GC tissues were SAPCD2-positive, compared with the 26.2% in the matched adjacent normal-appearing tissues (P<0.001). Furthermore, among the 121 gastritis tissues, SAPCD2 was overexpressed in precancerous gastric lesions such as dysplasia (Dys, 78.9%), intestinal metaplasia (IM, 44.7%), and chronic atrophic gastritis (CAG, 6.1%) compared with that in chronic non-atrophic gastritis (CNAG, 3.2%) (P<0.001). The SAPCD2-positivity rate was 81.3% in GC, suggesting that the expression of SAPCD2 increased with the severity of the lesion (P<0.001). In summary, we have described novel monoclonal antibodies against SAPCD2, which are highly expressed in GC tissues and may serve as the basis for an early clinical marker for GC development.

Long Noncoding RNAs-LET Behave as a Noncoding Signature for Early-Onset Menarche and Late-Onset Menopause in Breast Cancer Patients

Background: Breast cancer (BC) as a major cause of cancer-related death in women shows a very complex molecular and clinical phenotype, which has reduced the effectiveness of medical interventions. Evidence suggests that long noncoding RNAs (lncRNAs) are responsible for an important part of this complexity. This study aims to assess the expression and clinical implication of lncRNA LET in the pathobiology of BC. Materials and Methods: Quantitative real-time polymerase chain reaction was used to measure the expression of lncRNA-LET in breast tumors and adjacent normal-appearing tissues from 4 BC patients, as well as normal mammary tissues. Moreover, a bioinformatics approach was applied to uncover the potential lncRNA-LET-mediated sponge regulatory network as LET/miRNA/mRNA crosstalk. Results: Our study revealed that lncRNA-LET was significantly down-expressed not only in breast tumors but also in normal appearing breast tissues samples from BC subjects compared with true normal breast tissues obtained from healthy women. The low level of lncRNA-LET was meaningfully associated with early-onset menarche (≤13 years) and late-onset menopause (≥50) in patients. Moreover, the bioinformatics analyses support that lncRNA-LET could function as a tumor suppressor miRNA sponge. Conclusion: The results indicate that normal appearing breast tissues can undergo tumor-related molecular changes. Furthermore, they reveal the potential role of the dysregulation in LET-mediated ceRNA network in the pathophysiology of BC.

Mucosa\u2010associated cultivable aerobic gut bacterial microbiota among colorectal cancer patients attending at the referral hospitals of Amhara Regional State, Ethiopia

BackgroundColorectal cancer (CRC) is one of the top ten causes of cancer deaths in the world. Despite an increased prevalence of colorectal cancer has been documented from developing countries, there is no any report regarding gut microbiota among colorectal cancer patients in Ethiopia. Therefore, the current study evaluated cultivable aerobic gut bacterial distributions among malignant and its adjacent normal biopsies of CRC patients.MethodsCRC patients who were under colorectal cancer resection surgery during April 2017 to February 2018 at Felege Hiwot Referral and University of Gondar Teaching Hospitals enrolled in the study. Biopsy specimens were taken from malignant and its adjacent normal-appearing tissues. Bacterial cultivation, quantification and characterization of saline washed biopsies were performed under aerobic and candle jar conditions. Differences in bacterial microbiota compositions between malignant and normal tissue biopsies were evaluated and analyzed using Microsoft excel 2010 and GraphPad Prism5 statistical software.Results Fifteen CRC patients were participated with a mean age of 53.8 ± 10.8 years old and majorities (73.3 %) of patients were in between the age groups of 40 and 60 years old. The mean ± SD bacterial microbiota of malignant biopsies (3.2 × 105 ± 1.6 × 105 CFU/ml) was significantly fewer than that of adjacent normal tissue biopsies (4.0 × 105 ± 2.2 × 105 CFU/ml). This dysbacteriosis is positively correlated with the occurrence of CRC (p = 0.019). Proteobacteria (55.6 %), Firmicutes (33.3 %) and Fusobacteria (11.1 %) were the most frequently isolated phyla from non-malignant biopsies while only Proteobacteria (58.8 %) and Firmicutes (41.2 %) were from malignant ones. Family level differences were observed among phyla (Firmicutes and Proteobacteria) isolated from the study participants. For instance, the relative abundance of family Bacillaceae from malignant (26 %) was lower than the normal biopsies (39 %). On other hand, family Enterobacteriaceae was twice more abundant in malignant tissues (45 %) than in its matched normal tissues (23 %). Furthermore, the family Enterococcaceae (14 %) of phylum Firmicutes was solely isolated from malignant tissue biopsies.ConclusionsThe overall microbial composition of normal and malignant tissues was considerably different among the study participants. Further culture independent analysis of mucosal microbiota will provide detail pictures of microbial composition differences and pathogenesis of CRC in Ethiopian settings.

Abstract 1518: A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue

Abstract Lung adenocarcinoma (LUAD) is the most frequently diagnosed histological subtype of lung cancer and accounts for most smoking-related cancer deaths, warranting strategies for early intervention. Earlier work revealed genome-wide aberrations in LUADs and the adjacent premalignant field, known as “field carcinogenesis”, that are pertinent to LUAD pathogenesis. Yet, we still poorly understand the cellular and molecular architecture of LUAD and its nearby “field”. To fill this void, we performed, using the 10X Genomics system and NovaSeq 6000 platform, single-cell RNA sequencing (scRNA-seq) analysis of 4 early-stage resected LUADs as well as 11 matched normal lung tissues with differing spatial proximity from the tumors. We first analyzed 15,739 cells from a LUAD and matched tumor-adjacent and -distant normal tissues from patient 1. The resulting fraction of EPCAM+ cells from this analysis was approximately 4%. For deeper single-cell resolution of LUAD, we thus performed scRNA-seq of separately sorted/enriched epithelial cells (EPCAM+; n = 50,883) and non-epithelial cells (EPCAM-; n= 91,093) from patients 2, 3 and 4, each with an early-stage LUAD and three matched spatially-distributed normal-appearing tissues. Overall, we achieved on average approximately 150,000 reads and 1,956 genes detected per cell. Divergent populations of malignant and non-malignant cells and multiple epithelial and immune subsets clustered in an overall spatially modulated pattern according to proximity to the tumor. Hierarchical clustering revealed multiple distinct populations of airway lineage cells, including alveolar type 1 (AT1), alveolar type 2 (AT2), AT1/AT2 bipotential cells, club, goblet, basal and ciliated cells, with cells from the resected LUAD present in some but not all clusters. By analysis of epithelial-enriched fractions, we were also able to identify and interrogate rare cell subpopulations including CFTR-expressing ionocytes. By inference of copy number variation along with analysis of key oncogene expression patterns, we also identified epithelial cells that represent potential tumor cells-of-origin. Spatial reprogramming of field carcinogenesis encompassed immune cell populations including tumor-exclusive FOXP3+ regulatory T cells with marked over-expression of the major immune checkpoints CTLA4 and TIGIT. In contrast, for at least some of the cases, tumors were nearly devoid of natural killer cells, and abundance of CD8+ T cells dampened with increasing proximity to the tumor. Our single-cell surveys offer insights into novel cues in LUAD pathogenesis. Efforts are underway to interrogate the single-cell epithelial and immune landscape of additional LUADs and matched nearby normal-appearing lung, including those from non-smokers, to better understand the evolution of the disease and, thus, identify a low-hanging fruit of targets for early management of this fatal malignancy. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Patrick Brennan, Kieko Hara, Kyle Chang, Elena Bogatenkova, Beatriz Sanchez-Espiridion, Carmen Behrens, Jianjun Zhang, Boris Sepesi, Tina Cascone, Don L. Gibbons, Jichao Chen, George Eapen, Edwin J. Ostrin, Junya Fujimoto, Avrum E. Spira, Paul A. Scheet, Ignacio I. Wistuba, Linghua Wang, Humam Kadara. A single-cell transcriptomic atlas of lung adenocarcinoma and adjacent normal-appearing tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1518.

Upregulated circ RNA hsa_circ_0000337 promotes cell proliferation, migration, and invasion of esophageal squamous cell carcinoma.

BackgroundAs a new class of endogenous ncRNAs, circRNAs have been recently verified to be involved in the carcinogenesis and progression of human cancers. In the current study, we attempted to explore the potential function of a candidate circRNA (hsa_circ_0000337) in esophageal squamous cell carcinoma (ESCC).Patients and methodsThe altered expression of hsa_circ_0000337 was validated in clinical samples from 48 patients with ESCC. The human esophageal carcinoma cell lines KYSE-150 and TE-1, and the normal human esophageal epithelial cell line (HET-1A) were applied for functional analysis of hsa_circ_0000337. Cell proliferation was measured using the Cell Counting Kit-8 assay and the colony formation assay. Cell invasion and migration were detected by Transwell and wound healing assays, respectively. We further performed bioinformatic analysis and luciferase reporter assays to explore the role of hsa_circ_0000337 as a miRNA sponge.Resultshsa_circ_0000337 was significantly upregulated in ESCC tissues compared to adjacent normal-appearing tissues (P<0.0001). In our in vitro experiment, the expression of hsa_circ_0000337 was higher in TE-1 compared to the normal human esophageal epithelial cell line HET-1A (P<0.001), but was not significantly different in KYSE-150 (P>0.05). Knockdown of hsa_circ_0000337 significantly inhibited cell proliferation, migration, and invasion in TE-1 and KYSE-150 cell lines. Bioinformatics predicted and luciferase reporter assay verified that hsa_circ_0000337 could bind to miR-670-5p, a ncRNA involved in carcinogenesis. It is estimated that 21 genes are regulated by miR-670-5p.Conclusionhsa_circ_0000337 was found to be an upregulated circRNA that is related to ESCC and promotes the progression of disease by regulating cell proliferation, migration, and invasion. These findings suggest that this circRNA could be a promising diagnostic biomarker and potential therapeutic target.

Has_Circ_0030162: A Novel Epigenetic Biomarker and Potential Therapeutic Target for Esophageal Carcinoma

Background Circular RNA (circRNA), an endogenous non-coding RNA (ncRNA), plays a critical role in gene regulation and shows potential as diagnostic or prognostic biomarkers of human diseases. However, to date there has been little research on the role of circRNAs in the occurrence and development of esophageal cancer. Methods We adopted a three-stage design first using a high-throughput microarray chip to screen for differentially expressed circRNAs in esophageal squamous cell carcinoma (ESCC), then performing a validation study via quantitative reverse transcription polymerase chain reaction (qRT-PCR), and followed by functional analysis using in vitro experiments. In the discovery stage, we screened seven paired samples of ESCC and corresponding adjacent normal-appearing tissues for differentially expressed circRNAs. In the validation stage, we compared the expression of candidate circRNAs in 45 patients with ESCC and 45 healthy controls. In the functional analysis stage, we used three types of cell lines to explore the function of hsa_circ_0030162. Results In the discovery stage, we observed 459 up-regulated and 275 down-regulated differentially expressed circRNAs with FC ≥2.0 and P <0.05. Among these, hsa_circ_0030162 was ranked as the top down-regulated circRNA (FC=12.1). Thus, we selected this circRNA for validation using qRT-PCR and compared its expression in 45 patients with ESCC and 45 healthy controls. If using tissue hsa_circ_0030162 as the diagnostic biomarker, the AUC was 0.743 (95% CI: 0.639-0.848) with a sensitivity of 73.33% and a specificity of 66.67%. If using plasma hsa_circ_0030162 as the diagnostic biomarker, the AUC was 0.735 (95% CI: 0.632-0.837) with a sensitivity of 73.33% and a specificity of 62.22%. The in vitro experiments revealed that hsa_circ_0030162 could promote apoptosis and inhibit proliferation, migration, and invasion of ESCC cells. Luciferase reporter assays proved that it could bind to miR-125a-3p, a non-coding RNA involved in carcinogenesis. Conclusions Our results provide the first evidence that hsa_circ_0030162 plays an important role in the development and progression of ESCC. In addition, we illustrate the diverse roles of hsa_circ_0030162 in regulating cell proliferation, migration, invasion and apoptosis. Based upon our findings, we propose that hsa_circ_0030162 may act as a promising biomarker for clinical diagnosis of ESCC and suggest a new pathway for the development of anti-tumor therapeutics. Funding: The present study was supported by the National Natural Science Foundation of China [81673249], National Key R&361 D Program of China [2017YFC0907000], Social Development Project in Jiangsu Province [BE2015694], Scientific Research Innovation Project for Graduate Students in Jiangsu Province [KYCX17_1293], and Priority Academic Program Development of Jiangsu Higher Education Institutions PAAD]. Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by the Ethics Committee of Nanjing Medical University. After informed consents from all participants, questionnaires were used to collect demographic data.

Profiles of differentially expressed circRNAs in esophageal and breast cancer.

IntroductionCircular RNAs (circRNAs) function as efficient microRNA sponges with gene-regulatory potential and are promising cancer biomarkers. In this study, we used the Arraystar Human circRNA Array to construct a genome-wide circRNA profile of esophageal squamous cell cancer (ESCC) and breast cancer (BC).Patients and methodsExpression levels between cancer lesions and adjacent normal-appearing tissues were compared. We observed 469 upregulated circRNAs and 275 downregulated circRNAs in ESCC. Hsa_circRNA_103670 was upregulated 20.3-fold, while hsa_circRNA_030162 was downregulated 12.1-fold. For BC, 715 circRNAs were upregulated, and 440 circRNAs were downregulated. Hsa_circRNA_005230 was upregulated 12.2-fold, while hsa_circRNA_406225 was downregulated 12.4-fold.ResultsWhen we set the criteria as fold change in expression ≥2 between cancer and adjacent normal-appearing tissue with a P-value <0.01, there were 22 common circRNAs (11 upregulated and 11 downregulated) in relation to both ESCC and BC. Gene ontology and the Kyoto encyclopedia of genes and genomes analyses showed that these circRNAs were involved in the tumorigenesis of human cancers.ConclusionOur study revealed that circRNAs are promising candidates as valuable biomarkers for ESCC and BC, although relevant research is still in its infancy and the functional role of specific circRNAs in tumorigenesis is just starting to be elucidated.

The aberrant splicing of BAF45d links splicing regulation and transcription in glioblastoma.

Glioblastoma, the most aggressive primary brain tumor, is genetically heterogeneous. Alternative splicing (AS) plays a key role in numerous pathologies, including cancer. The objectives of our study were to determine whether aberrant AS could play a role in the malignant phenotype of glioma and to understand the mechanism underlying its aberrant regulation. We obtained surgical samples from patients with glioblastoma who underwent 5-aminolevulinic fluorescence-guided surgery. Biopsies were taken from the tumor center as well as from adjacent normal-appearing tissue. We used a global splicing array to identify candidate genes aberrantly spliced in these glioblastoma samples. Mechanistic and functional studies were performed to elucidate the role of our top candidate splice variant, BAF45d, in glioblastoma. BAF45d is part of the switch/sucrose nonfermentable complex and plays a key role in the development of the CNS. The BAF45d/6A isoform is present in 85% of over 200 glioma samples that have been analyzed and contributes to the malignant glioma phenotype through the maintenance of an undifferentiated cellular state. We demonstrate that BAF45d splicing is mediated by polypyrimidine tract-binding protein 1 (PTBP1) and that BAF45d regulates PTBP1, uncovering a reciprocal interplay between RNA splicing regulation and transcription. Our data indicate that AS is a mechanism that contributes to the malignant phenotype of glioblastoma. Understanding the consequences of this biological process will uncover new therapeutic targets for this devastating disease.

Secretory phospholipase-A2 and fatty acid composition in oral reactive lesions: a cross-sectional study

BackgroundOral reactive lesions are the most common lesions of oral cavity. Phospholipases and fatty acids play key roles in the creation of inflammation by change in metabolic activities and production of lipid mediators. The aim of this study was to investigate the amount of secretory phospholipase-A2 (sPLA2) and difference of fatty acid pattern in oral reactive hyperplasia and adjacent normal appearing tissues in patients with oral reactive lesions.MethodsPaired samples of oral hyperplastic (OH) and adjacent normal-appearing tissue of 45 patients were investigated in this study. The collected samples were analyzed with enzymatic spectrophotometric method in terms of the amount of sPLA2 and composition of fatty acids by gas–liquid chromatography method.ResultsThe amount of sPLA2 (1.8-fold, p < 0.001), stearic acid (1.2-fold, p < 0.001), oleic acid (1.1-fold, p = 0.01), arachidonic acid (1.5-fold, p < 0.001) and docosahexaenoic acid (1.3-fold, p = 0.02) were increased, while the amount of palmitoleic acid (−45%, p < 0.001) and linoleic acid (−19%, p < 0.001) were reduced in the OH tissue samples. Furthermore, the results demonstrated significant associations between the type and location of tissue samples with monounsaturated fatty acids (MUFAs) and n−3 polyunsaturated fatty acids. Tissue samples from patients with inflammatory fibroepithelial hyperplasia showed relatively higher MUFAs and lower n−3 polyunsaturated fatty acids than other type of lesions.ConclusionsLocalized changes in the sPLA2 activity and composition of fatty acid are associated with oral reactive hyperplasia and the type of pathological response. We suggest that sPLA2 activity and multiple type of fatty acids might be used as potential therapeutic target for oral reactive hyperplasia.

Abstract A51: DNA methylation in tumor, adjacent non-tumorous and normal breast tissues

Abstract DNA methylation is an essential epigenetic mechanism involved in gene regulation. Aberrant DNA methylation plays an important role in breast cancer initiation and progression via inactivation of tumor suppressor genes and activation of oncogenes. Conventional approaches comparing DNA methylation profiles in tumor and adjacent non-tumorous tissue have shown that changes in DNA methylation contribute to deregulation of gene expression in breast cancer. However, adjacent non-tumorous tissue represents a suboptimal baseline control because its molecular profile may have been altered, and to some extent, similar to that of tumor. Consequently, important breast cancer-associated DNA methylation may not be identifiable in such comparisons. Using RNA-sequencing data generated from freshly-frozen breast tissue samples, we examined expression of methylation-related genes in 119 breast tumor, 47 adjacent normal-appearing breast tissue samples, and 23 normal breast tissue samples from healthy donors. Bowtie and RESM algorithms were used to align raw reads and quantify transcript abundance. Differential expression analyses were performed using edgeR software and normalized transcript counts. Normal breast tissue from healthy women compared to tumor from cancer patients identified differentially expressed cancer genes that are methylated in tumor, such as BRCA1, ESR1, ESR2, CDKN2A, HIC1, CDCP1, and SFRP1. A subset of these genes were found to be deregulated in adjacent non-tumorous tissue in a similar way as in tumor tissue, including PTGS2, SFN, SYK, FHIT , CST6, GADD45A, and THBS1, and are thus undetectable in the comparison between tumor and adjacent non-tumor tissue. In addition, a set of methylation-related genes were identified only in the comparison between tumor and normal tissue, including genes directly involved in the methylation/epigenetic process (HIST1H4A, KDM1B, MAT2B and SAP18) as well as novel target genes that may be methylated and play an important role in carcinogenesis (NFIA, ARHGAP18, and GRHL2). These findings suggest that normal breast tissue from healthy women is the most desirable control in comparison with tumor tissue to identify novel, tissue-specific methylation changes that may be important for carcinogenesis. Citation Format: Erin Wagner, Wenting Wu, Yunlong Liu, Kenneth Nephew, Jiali Han, Chunyan He. DNA methylation in tumor, adjacent non-tumorous and normal breast tissues. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A51.

Tissue fatty acid composition and secretory phospholipase-A2 activity in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a remarkable health problem worldwide, but its pathogenesis remains unknown. The aim of this study was to compare fat composition and secretory phospholipase-A2 (sPLA2) activity between the malignant and adjacent normal squamous tissues in patients with OSCC. Paired samples of malignant squamous and adjacent normal-appearing tissues were collected from 27 patients with OSCC. The fatty acid composition in the obtained tissues was determined by gas liquid chromatography. Tissue enzyme activities of sPLA2 were measured using the standard assay with Diheptanoyl Thio-Phosphatidylcholine as substrate. In the OSCC tissue, the level of stearic acid (18:0) and activity of sPLA2 were higher (P < 0.001), and the levels of oleic acid (18:1n-9) and linoleic acid (18:2n-6) were lower than that in the adjacent normal-appearing squamous tissue (P < 0.001). The activity of sPLA2 in OSCC was strongly negatively correlated with the amount of 18:2n-6 (r = -0.41, P < 0.001). Negative significant associations were observed between the OSCC invasion and tissue levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHE). The changes in the fatty acid composition and sPLA2 activity may be regarded as indicators of altered lipid metabolism occurring in vivo during squamous cell carcinogenesis.

Abstract 3268: Differences in expression of lncRNAs in breast tumor, adjacent normal-appearing breast tissue, and normal breast tissue from healthy donors

Abstract Long noncoding RNAs (lncRNAs) are an emerging class of key regulatory RNAs that are greater than 200nt and do not code for protein. They appear to be critical for various biological processes including gene regulation. Increasing evidence has suggested that aberrantly expressed lncRNAs play a key role in the initiation and progression of breast cancer. In most studies of breast cancer, breast tumor tissue was compared to adjacent normal-appearing tissue to identify differentially expressed lncRNAs. However, expression of lncRNAs in normal breast tissue from healthy donors has rarely been studied, and the differences in expression of lncRNAs in breast tumor and normal breast tissue from healthy donors remain largely unknown. Using RNA-sequencing data generated from freshly-frozen breast tissue samples, we examined the expression of lncRNAs in 119 breast tumor, 47 adjacent normal-appearing breast tissue samples, and 23 normal breast tissue samples from healthy donors. Bowtie and RESM algorithms were used to align raw reads and quantify transcript abundance. Differential expression analyses were performed using edgeR software. We assessed the expression of a total of 7,844 mapped lncRNAs using normalized transcript counts. Unsupervised Principal Components Analysis (PCA) demonstrated that the lncRNA expression profile of adjacent normal-appearing tissue was different compared to normal breast tissue from healthy donors, and partially overlapped with the tumor. At various fold change thresholds, more differentially expressed lncRNAs can be detected when the tumor tissue is compared to normal tissue than to adjacent non-tumorous tissue. With a false discovery rate (FDR) &amp;lt; 0.05 and a fold change (FC) of more than ±2, we identified 107 differentially expressed lncRNAs for the tumor vs. normal breast tissue, 53 lncRNAs for tumor vs. adjacent normal-appearing tissue, and 20 lncRNAs for adjacent normal-appearing vs. normal breast tissue. In our analyses of tumor vs. adjacent normal-appearing tissue and tumor vs. normal tissue from healthy donors, we identified differentially expressed lncRNAs including HOTAIR, GAS5 and ANRIL/CDKN2B-AS1 that were previously implicated in breast cancer, with considerably higher fold changes in latter comparison. When using normal breast tissue from healthy donors as the baseline, we identified novel putative breast cancer-associated lncRNAs including EMX2OS, TERC, HOTAIRM1, and WT1-AS that are aberrantly expressed in breast tumor. These lncRNAs were not identifiable in a direct comparison of tumor and adjacent normal-appearing tissue. Our results suggest that adjacent normal-appearing tissue undergoes changes in lncRNAs expression similar to those observed in tumor tissue to some extent, and a number of new and potentially important lncRNAs can only be detected using normal tissue from healthy donors as an optimal baseline tissue. Citation Format: Erin Wagner, Yunlong Liu, Bryan Schneider, Anna Maria Storniolo, Jiali Han, Chunyan He. Differences in expression of lncRNAs in breast tumor, adjacent normal-appearing breast tissue, and normal breast tissue from healthy donors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3268. doi:10.1158/1538-7445.AM2014-3268

Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition.

PurposeStearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue.MethodsPaired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively.ResultsBreast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 µM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant.ConclusionThe fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition may also, in addition to Δ9 desaturation, modulate other reactions in de novo fatty acid synthesis and lipogenesis, and subsequently affect the overall survival and progression of breast cancer.

Evidence for a field effect in early prostate cancer (PCa): Gene expression profiles in normal-appearing prostate tissue (NT) adjacent to tumor (T) as predictors of clinical outcome.

5029 Background: We previously identified genes whose expression predicts aggressive PCa (clinical recurrence (cR), prostate cancer death (PCD), adverse pathology) when assessed in histologically heterogeneous tumor foci and in biopsies (Klein ASCO 2012). These results enabled the definition of a multi-gene Genomic Prostate Score (GPS), which has been clinically validated (Cooperberg AUA 2013). There is interest regarding a possible field effect in PCa, i.e. molecular alterations throughout the gland that may influence PCa development. We conducted exploratory analyses to evaluate gene expression, including GPS, in adjacent normal-appearing tissue (NT) for prediction of cR and PCD. Methods: Cohort sampling was used to select 127 patients with and 374 without cR from 2,641 patients treated with RP for T1/T2 PCa. Expression of 732 genes was measured by qRT-PCR separately in T and NT (defined as &gt; 3 mm from T) specimens. GPS (0-100 units) was determined using the genes and algorithm from the validation study. Analysis used Cox proportional hazards models and Storey’s false discovery rate (FDR) control. Results: 410 evaluable patients had paired T and NT. Of the 405 genes which were predictive of outcome in T (FDR &lt; 20%), 289 (71%) showed similar but weaker effects in NT. 47 genes were associated with cR in NT (FDR &lt; 20%), of which 34 also concordantly predicted cR in T (FDR &lt; 20%). GPS assessed in NT significantly predicted time to cR (HR/20 units = 1.8; 95% CI: 1.3-2.4; p&lt; 0.001) and PCD (HR/20 units = 1.9; 95% CI: 1.2-3.0; p = 0.005) but was less predictive than GPS in T (HR/20 units = 4.8 for cR; 95% CI: 3.7-6.2; p &lt; 0.001 and HR/20 units = 6.9 for PCD; 95% CI: 4.4-10.7; p &lt; 0.001). The strongest components of GPS in predicting cR and PCD in NT were stromal response and androgen signaling genes (p &lt; 0.05); proliferation and cellular organization genes did not consistently provide a significant contribution in NT. Conclusions: These data indicate that gene expression profiles, including GPS, can predict outcome in NT, albeit more weakly than in tumor. These findings suggest that there is an underlying field effect associated with the development of aggressive PCa.

Plasma Cytokines as Potential Response Indicators to Dietary Freeze-Dried Black Raspberries in Colorectal Cancer Patients

Oral consumption of freeze-dried black raspberries attenuated neoplastic changes in colorectal tissue markers of apoptosis, cell proliferation, and angiogenesis in colorectal cancer (CRC) patients. To determine whether plasma concentrations of interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, granulocyte macrophage colony stimulating factor (GM-CSF), interferon-γ, and tumor necrosis factor-α (TNF-α) were associated with berry treatment and changes in colorectal tissue markers of apoptosis, cell proliferation, and angiogenesis, plasma and biopsy samples of adenocarcinoma and adjacent normal-appearing colorectal tissue were collected before and during berry treatment from 24 CRC patients who had not received prior therapy and drank a slurry of black raspberry powder (20 g in 100 ml drinking water) 3 times a day for 1 to 9 wk. Plasma concentrations of GM-CSF (+0.12 ± 0.04 pg/mL; P = 0.01) and IL-8 (-1.61 ± 0.71 pg/mL; P = 0.04) changed in patients receiving berries for more than 10 days. These changes were correlated with beneficial changes in markers of proliferation (rΔGM-CSF, ΔKi67 carcinoma - normal = −0.51) and apoptosis (rΔIL-8, ΔTUNEL carcinoma - normal = −0.52) observed in colorectal tissue taken within the same week. Plasma concentrations of GM-CSF and IL-8 may serve as noninvasive indicators to monitor tissue response to berry-based interventions for CRC.

Genome-wide identification of OTP gene as a novel methylation marker of breast cancer

Aberrant DNA methylation occurs early and frequently in tumorigenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the clinical process of breast cancer diagnosis. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with high-resolution CpG microarray analysis to identify hypermethylated genes in breast cancer. Among differentially methylated genes between tumor and adjacent normal tissues, 3 candidate genes (LHX2, WT1 and OTP) were finally selected through a step-wise filtering process and examined for methylation status in normal tissues, primary tumor, and paired adjacent normal-appearing tissues from 39 breast cancer patients. Based on the calculated cut-off values, all genes showed significantly higher frequencies of aberrant hypermethylation in primary tumors (43.6% for LHX2, 89.7% for WT1 and 100% for OTP, p<0.05) while frequencies were intermediate in paired adjacent normal tissues and absent in normal tissues. On further analysis, the methylation level in primary tumors was not significantly correlated with clinicopathological features. Interestingly, DNA methylation of a novel gene OTP was detected in adjacent normal tissues even 6cm away from primary tumors, suggesting that OTP methylation may qualify as a biomarker for the early detection of breast cancer. In conclusion, we successfully identified a novel gene OTP frequently methylated in breast cancer by genome-wide screening. Our results suggest that the OTP gene may play a crucial role in breast carcinogenesis, although further clinical validation will be needed to evaluate the potential application of OTP in the early detection of breast cancer.

Promoter hypermethylation of RASSF1A, MGMT, and HIC-1 genes in benign and malignant colorectal tumors

Hypermethylation at the promoter region is an important epigenetic mechanism underlying the inactivation of tumor suppressor genes and frequently occurs as an early event in the development of different types of cancer including colorectal carcinoma (CRC). The aim of the present study is the detection of methylation status for some tumor suppressor genes including RASSF1A, MGMT, and HIC-1 in both cancerous and precancerous lesions of colorectal mucosa to evaluate the possibility of developing epigenetic biomarker for early detection of Egyptian CRC. Tissue biopsy was collected from 72 patients (36 CRC, 17 adenomatous polyps, and 19 ulcerative colitis), and in addition, adjacent normal-appearing tissues were collected as control. Promoter hypermethylation status for RSSAF1A, MGMT, and HIC-1 genes was detected after isolation of genomic DNA from the tissues samples using methylation-specific PCR technique. High frequency of methylation at MGMT, RASSFA, and HIC-1 was detected in CRC patients (25%, 47.2%, and 41.7% respectively). The highest methylation detected in adenomatous polyps patients was in MGMT gene (47.1%) followed by 35.3% for HIC-1 and only 5.9% for RASSF1A gene. HIC-1 gene exhibited highest frequency of methylation in ulcerative colitis patients (57.8%) whereas it was 26.3% for both RASSF1A and MGMT genes. A nonsignificant association was recorded between the methylation status in different genes examined with the clinicopathological factors except the association between methylation at RASSF1A gene with gender (p=0.005), and it was significant. In conclusion, aberrant hypermethylation at promoter region of RASSFA, MGMT, and HIC-1 genes is involved in Egyptian CRCs. Hypermethylation of MGMT and HIC-1 genes plays an important role in the initiation of disease especially ulcerative colitis-carcinoma pathway.

Mitochondrial Prohibitins and Septin 9 Are Implicated in the Onset of Rat Hepatocarcinogenesis

In the present study, protein lysates from microdissected glutathione S-transferase placental-form-positive (GST-P(+)) foci and hepatocellular carcinomas from livers of rats treated with N-diethylnitrosamine followed by phenobarbital at doses of 0 and 500 ppm in the diet for 10 and 33 weeks were analyzed using QSTAR Elite liquid chromatography with tandem mass spectrometry and iTRAQ technology. Among 75 proteins, a total of 27 and 50 proteins displaying significant quantitative changes comparing with adjacent normal-appearing liver tissue were identified in GST-P(+) foci of initiation control and promotion groups, respectively, which are related to transcription, protein folding, cytoskeleton filaments reorganization, cell cycle control, nuclear factor (erythroid-derived 2)-like 2 (NRF2)-mediated oxidative stress responses, lipid metabolism, glutathione metabolism, oxidative phosphorylation, and signal transduction. Furthermore, Ingenuity Pathway and bioinformatic analyses revealed that expression changes of genes encoding proteins with altered expression detected in GST-P(+) foci are likely to be controlled by c-myc, NRF2, aryl hydrocarbon receptor, nuclear factor kappa B, and hepatocyte nuclear factor 4 transcriptional factors. Coordinated overexpression of mitochondrial chaperons prohibitin (PHB) and prohibitin 2 (PHB2), septin 9 (SEPT9), neurabin 1, and other cytoskeletal and functional proteins in areas of GST-P(+) foci during initiation and/or promotion stages of rat hepatocarcinogenesis was associated with induction of cell proliferation and might be responsible for the neoplastic transformation of rat liver preneoplastic lesions. Newly discovered elevation of PHB, PHB2, and SEPT9 in GST-P(+) foci and tumors, imply that they might play important role in the onset of liver cancer and be of potential values in the studies of hepatocarcinogenesis.