Cell-Fiber-Based 3D Tissue Array for Drug Response Assay

For the establishment of a reproducible and sensitive assay system for three-dimensional (3D) tissue-based drug screening, it is essential to develop 3D tissue arrays with uniform shapes and high-cell numbers that prevent cell death in the center of the tissue. In recent years, 3D tissue arrays based on spheroids have attracted increased attention. However, they have only been used in specific tissues with hypoxic regions, such as cancer tissues, because nutrient deprivation and hypoxic regions are formed in the core as spheroids grow. Herein we propose a method to array cell-encapsulated tube-like tissue (cell fiber (CF)) with diameters <150 µm to prevent nutrient deprivation and hypoxia using a device that can fix the CFs, section them in uniform sizes and transfer them to a 96-well plate. We fabricated the arrays of CF fragments from cell lines (GT1-7), cancer cells (HeLa), mouse neural stem cells (mNSCs), and differentiated mNSCs, and performed drug response assays. The array of CF fragments assessed drug response differences among different cell types and drug responses specific to 3D tissues. The array of CF fragments may be used as a versatile drug screening system to detect drug sensitivities in various types of tissues.

The Identification of RNA Modification Gene PUS7 as a Potential Biomarker of Ovarian Cancer

RNA modifications are reversible, dynamically regulated, and involved in a variety of diseases such as cancers. Given the lack of efficient and reliable biomarkers for early diagnosis of ovarian cancer (OV), this study was designed to explore the role of RNA modification genes (RMGs) in the diagnosis of OV. Herein, 132 RMGs were retrieved in PubMed, 638 OV and 18 normal ovary samples were retrieved in The Cancer Genome Atlas (TCGA), and GSE18520 cohorts were collected for differential analysis. Finally, PUS7 (Pseudouridine Synthase 7) as differentially expressed RMGs (DEGs-RMGs) was identified as a diagnostic biomarker candidate and evaluated for its specificity and sensitivity using Receiver Operating Characteristic (ROC) analysis in TCGA and GEO data. The protein expression, mutation, protein interaction networks, correlated genes, related pathways, biological processes, cell components, and molecular functions of PUS7 were analyzed as well. The upregulation of PUS7 protein in OV was confirmed by the staining images in HPA and tissue arrays. Collectively, the findings of the present study point towards the potential of PUS7 as a diagnostic marker and therapeutic target for ovarian cancer.

The role of opiorphin genes in prostate cancer growth and progression

Background: We describe the first studies investigating a role for opiorphin genes ( PROL1, SMR3A and SMR3B) in prostate cancer (PrCa). Materials & methods: Databases and PrCa tissue arrays were screened for opiorphin expression. Xenografted tumor growth of human PrCa cells overexpressing PROL1 was compared with controls in nude mice. Modulated gene expression by overexpression of PROL1 was determined by RNA sequencing. Results: PrCa is associated with overexpression of opiorphin genes. Xenografted androgen-sensitive PrCa cells overexpressing PROL1 developed into tumors in castrated male mice (in contrast to parental cells). PROL1 overexpression modulates expression of genes in angiogenesis, steroid and hypoxic response pathways. Conclusions: Opiorphins promote the development of androgen-insensitive PrCa and activate pathways that potentially overcome the hypoxic barrier generated during tumor growth.

Aberrantly Expressed RECQL4 Helicase Supports Proliferation and Drug Resistance of Human Glioma Cells and Glioma Stem Cells

Anti-tumour therapies eliminate proliferating tumour cells by induction of DNA damage, but genomic aberrations or transcriptional deregulation may limit responses to therapy. Glioblastoma (GBM) is a malignant brain tumour, which recurs inevitably due to chemo- and radio-resistance. Human RecQ helicases participate in DNA repair, responses to DNA damage and replication stress. We explored if a helicase RECQL4 contributes to gliomagenesis and responses to chemotherapy. We found upregulated RECQL4 expression in GBMs associated with poor survival of GBM patients. Increased levels of nuclear and cytosolic RECQL4 proteins were detected in GBMs on tissue arrays and in six glioma cell lines. RECQL4 was detected both in cytoplasm and mitochondria by Western blotting and immunofluorescence. RECQL4 depletion in glioma cells with siRNAs and CRISPR/Cas9 did not affect basal cell viability, slightly impaired DNA replication, but induced profound transcriptomic changes and increased chemosensitivity of glioma cells. Sphere cultures originated from RECQL4-depleted cells had reduced sphere forming capacity, stronger responded to temozolomide upregulating cell cycle inhibitors and pro-apoptotic proteins. RECQL4 deficiency affected mitochondrial network and reduced mitochondrial membrane polarization in LN18 glioblastoma cells. We demonstrate that targeting RECQL4 overexpressed in glioblastoma could be a new strategy to sensitize glioma cells to chemotherapeutics.

Investigating the role of opiorphin genes in prostate cancer and the possible genetic mechanisms by which they modulate tumor growth and androgen-sensitivity.

Background The opiorphin family of genes (represented in humans by ProL1, hSMR3A and hSMR3B) encode peptides which act as potent neutral endopeptidase (NEP) inhibitors. Because modulated NEP activity is associated with cancer development it has been suggested that dysregulated opiorphin expression may be involved in oncogenesis and recent reports have associated upregulated opiorphin gene expression with breast and oropharyngeal cancer. These observations prompted the present studies to determine if opiorphin genes play a role in prostate cancer (PrCa).Methods Publicly available data bases were screened for evidence associating opiorphin gene expression with PrCa and the findings confirmed using PrCa tissue arrays. Androgen-insensitive (PC3) and -sensitive (LNCaP) PrCa cells were engineered to overexpress ProL1, referred to as LNCaP-ProL1+ and PC3-ProL1+, respectively. Xenografted tumor growth of ProL1-overexpressing and parental cell-lines were compared in male, castrated-male and female nude mice. Changes in global gene expression resulting from overexpression of ProL1 in these cell-lines was determined by RNA-Seq.Results Publicly available datasets supported an association between overexpression of opiorphin genes and PrCa, which was confirmed using tissue arrays. Xenografted tumors derived from PC3-ProL1+ had an initial growth advantage over parent cell-lines in male mice, although at later time points there was no difference. Xenografted tumors derived from LNCaP-ProL1+ were able to grow in castrated male mice (in contrast to the parent cell-lines), and had impaired growth in female mice. Global gene expression analysis demonstrated that overexpression of ProL1 causes modulated expression of genes involved in signaling, angiogenesis and steroid response pathways.Conclusions This is the first report associating upregulated opiorphin gene expression and PrCa. Xengrafted tumors derived from the androgen-sensitive LNCaP cell-line engineered to overexpress ProL1 exhibit a more androgen-insensitive phenotype. Previous reports demonstrate opiorphins act as master regulators of the hypoxic response in smooth muscle cells. RNAseq data not only supports a similar role for ProL1 in PrCa (regulating genes involved in angiogenesis), but in addition, regulating genes involved in steroid response. Therefore, opiorphins may play a role in PrCa development by activating pathways that overcome the hypoxic environment of the developing tumor and promote the development of androgen-insensitivity.

Investigating the role of opiorphin genes in prostate cancer and the possible genetic mechanisms by which they modulate tumor growth and androgen-sensitivity.

Background The opiorphin family of genes (represented in humans by ProL1, hSMR3A and hSMR3B) encode peptides which act as potent neutral endopeptidase (NEP) inhibitors. Because modulated NEP activity is associated with cancer development it has been suggested that dysregulated opiorphin expression may be involved in oncogenesis and recent reports have associated upregulated opiorphin gene expression with breast and oropharyngeal cancer. These observations prompted the present studies to determine if opiorphin genes play a role in prostate cancer (PrCa). Methods Publicly available data bases were screened for evidence associating opiorphin gene expression with PrCa and the findings confirmed using PrCa tissue arrays. Androgen-insensitive (PC3) and -sensitive (LNCaP) PrCa cells were engineered to overexpress ProL1 , referred to as LNCaP-ProL1+ and PC3-ProL1+, respectively. Xenografted tumor growth of ProL1-overexpressing and parental cell-lines were compared in male, castrated-male and female nude mice. Changes in global gene expression resulting from overexpression of ProL1 in these cell-lines was determined by RNA-Seq. Results Publicly available datasets supported an association between overexpression of opiorphin genes and PrCa, which was confirmed using tissue arrays. Xenografted tumors derived from PC3-ProL1+ had an initial growth advantage over parent cell-lines in male mice, although at later time points there was no difference. Xenografted tumors derived from LNCaP-ProL1+ were able to grow in castrated male mice (in contrast to the parent cell-lines), and had impaired growth in female mice. Global gene expression analysis demonstrated that overexpression of ProL1 causes modulated expression of genes involved in signaling, angiogenesis and steroid response pathways. Conclusions This is the first report associating upregulated opiorphin gene expression and PrCa. Xengrafted tumors derived from the androgen-sensitive LNCaP cell-line engineered to overexpress ProL1 exhibit a more androgen-insensitive phenotype. Previous reports demonstrate opiorphins act as master regulators of the hypoxic response in smooth muscle cells. RNAseq data not only supports a similar role for ProL1 in PrCa (regulating genes involved in angiogenesis), but in addition, regulating genes involved in steroid response. Therefore, opiorphins may play a role in PrCa development by activating pathways that overcome the hypoxic environment of the developing tumor and promote the development of androgen-insensitivity.

Immunohistochemical staining reveals differential expression of ACSL3 and ACSL4 in hepatocellular carcinoma and hepatic gastrointestinal metastases

Long-chain fatty acyl CoA synthetases (ACSLs) activate fatty acids by CoA addition thus facilitating their intracellular metabolism. Dysregulated ACSL expression features in several cancers and can affect processes such as ferroptosis, fatty acid β-oxidation, prostaglandin biosynthesis, steroidogenesis and phospholipid acyl chain remodelling. Here we investigate long chain acyl-CoA synthetase 3 (ACSL3) and long chain acyl-CoA synthetase 4 (ACSL4) expression in liver malignancies. The expression and subcellular localisations of the ACSL3 and ACSL4 isoforms in hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA) and hepatic metastases were assessed by immunohistochemical analyses of multiple tumour tissue arrays and by subcellular fractionation of cultured HepG2 cells. The expression of both enzymes was increased in HCC compared with normal liver. Expression of ACSL3 was similar in HCC and hepatic metastases but lower in healthy tissue. Increased ACSL3 expression distinguished HCC from CCA with a sensitivity of 87.2% and a specificity of 75%. ACSL4 expression was significantly greater in HCC than in all other tumours and distinguished HCC from normal liver tissue with a sensitivity of 93.8% and specificity of 93.6%. Combined ACSL3 and ACSL4 staining scores distinguished HCC from hepatic metastases with 80.1% sensitivity and 77.1% specificity. These enzymes had partially overlapping intracellular distributions, ACSL4 localised to the plasma membrane and both isoforms associated with lipid droplets and the endoplasmic reticulum (ER). In conclusion, analysis of ACSL3 and ACSL4 expression can distinguish different classes of hepatic tumours.

HMGCS2 Mediates Ketone Production and Regulates the Proliferation and Metastasis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor worldwide; however, the traditional therapeutic approaches and survival rates are still limited. To improve current therapies, it is necessary to investigate the molecular mechanisms underlying liver cancer and to identify potential therapeutic targets. The aims of this study were to verify the mechanisms and therapeutic potential of the ketogenesis rate-limiting enzyme 3-Hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in HCC. Immunohistochemical staining of human liver disease tissue arrays showed that HMGCS2 is abundantly expressed in normal liver tissues but is downregulated in cirrhosis and HCC tissues. In HCC patients, lower HMGCS2 expression was correlated with higher pathological grades and clinical stages. In our investigation of the molecular mechanisms of HMGCS2 in HCC, we showed that knockdown of HMGCS2 decreased ketone production, which promoted cell proliferation, cell migration, and xenograft tumorigenesis by enhancing c-Myc/cyclinD1 and EMT signaling and by suppressing the caspase-dependent apoptosis pathway. Ketone body treatment reduced the proliferation- and migration-promoting effects of HMGCS2 knockdown in cells. In contrast, HMGCS2 overexpression increased the intracellular ketone level and inhibited cell proliferation, cell migration, and xenograft tumorigenesis. Finally, ketogenic diet administration significantly inhibited liver cancer cell growth in mice. Our studies highlight the potential therapeutic strategy of targeting HMGCS2-mediated ketogenesis in liver cancer.

Investigating the role of opiorphin genes in prostate cancer and the possible genetic mechanisms by which they modulate tumor growth and androgen-sensitivity

Background The opiorphin family of genes (represented in humans by ProL1, hSMR3A and hSMR3B) encode peptides which act as potent neutral endopeptidase (NEP) inhibitors. Because modulated NEP activity is associated with cancer development, several years ago a review postulated that dysregulated opiorphin expression may be involved in oncogenesis. This has recently been supported by reports associating changes in expression of opiorphin-encoding genes with breast and oropharyngeal cancer. Based on these reports, we initiated the studies described here to determine if opiorphin-encoding genes play a role in prostate cancer (PrCa).Methods We screened publicly available data bases for evidence of an association between PrCa and opiorphin gene expression and then confirmed these findings using PrCa tissue arrays. Cell-lines representing early (LNCaP) and late stage (PC3) PrCa were engineered to overexpress ProL1 and the growth of xenografted tumors from these lines compared to parent cell lines in male and female nude mice. The changes in global gene expression caused by overexpression of ProL1 in these cell lines was determined by RNA-Seq to identify possible genetic mechanisms related to growth and progression of PrCa.Results Two datasets available on GEO showed an association between upregulation of opiorphin-encoding genes and PrCa, which was confirmed using tissue arrays. Xenografted tumors derived from PC3 engineered to overexpress ProL1 had an initial growth advantage over parent cell lines whilst tumors derived from LNCaP cells overexpressing ProL1 had impaired growth in female mice. Global gene expression analysis suggested that overexpression of ProL1 causes modulated expression of genes involved in signaling, angiogenesis and steroid response pathways. These pathways might be involved in overcoming the hypoxic barrier of the growing tumor and the development of androgen insensitivity.Conclusions This is the first report to show an association between upregulation of opiorphin-encoding genes and PrCa and that their overexpression in PrCa cell-lines modulates tumor growth in mouse xenograft models. Overexpression of opiorphin-encoding genes modulates genetic pathways that previous reports have associated with PrCa growth, suggesting possible genetic mechanisms for tumor development.