Serum microRNA-29a is a promising novel marker for early detection of colorectal liver metastasis

Background: Colorectal cancer (CRC) metastasis occurs in various organs, most frequently in liver. Serological examination including tumor and biochemical markers for liver function evaluation is routinely performed, though its accuracy is not high. MicroRNAs (miRNAs) have been implicated in a variety of human diseases including cancer, and have many characteristics of an ideal biomarker most notably their inherent stability and resilience. Recently, several studies have indicated that circulating miRNAs hold much potential as novel noninvasive biomarkers for cancer and other disease processes. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for CRC with liver metastasis. Methods: This study was divided into three phases: (I) 3 candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 38 CRC patients with liver metastasis and 36 CRC patients without metastasis. (II) Marker validation by real-time RT-PCR on a similar cohort of age- and sex-matched CRC patients without (n = 20) and with liver metastasis (n = 20). (III) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of CRC patients. Results: Serum miR-29a was significantly higher in colorectal liver metastasis (CRLM) patients than in CRC patients. This marker yielded a receiver operating characteristic curve area of 80.3%. At a cutoff value of 0.155, the sensitivity was 75% and the specificity was 75% in discriminating metastatic from non-metastatic patients. In addition, increased levels of miR-29a expression were also observed in colorectal tumors from CRLM patients compared with CRC patients. No significant difference was observed in the levels of serum miR-92a between metastatic and non-metastatic patients. Conclusions: These findings suggest that serum miR-29a has strong potential as a novel noninvasive biomarker for early detection of CRC with liver metastasis.     

Gene expression signature analysis identifies vorinostat as a candidate therapy for gastric cancer

Background

Gastric cancer continues to be one of the deadliest cancers in the world and therefore identification of new drugs targeting this type of cancer is thus of significant importance. The purpose of this study was to identify and validate a therapeutic agent which might improve the outcomes for gastric cancer patients in the future.

Methodology/Principal Findings

Using microarray technology, we generated a gene expression profile of human gastric cancer–specific genes from human gastric cancer tissue samples. We used this profile in the Broad Institute''s Connectivity Map analysis to identify candidate therapeutic compounds for gastric cancer. We found the histone deacetylase inhibitor vorinostat as the lead compound and thus a potential therapeutic drug for gastric cancer. Vorinostat induced both apoptosis and autophagy in gastric cancer cell lines. Pharmacological and genetic inhibition of autophagy however, increased the therapeutic efficacy of vorinostat, indicating that a combination of vorinostat with autophagy inhibitors may therapeutically be more beneficial. Moreover, gene expression analysis of gastric cancer identified a collection of genes (ITGB5, TYMS, MYB, APOC1, CBX5, PLA2G2A, and KIF20A) whose expression was elevated in gastric tumor tissue and downregulated more than 2-fold by vorinostat treatment in gastric cancer cell lines. In contrast, SCGB2A1, TCN1, CFD, APLP1, and NQO1 manifested a reversed pattern.

Conclusions/Significance

We showed that analysis of gene expression signature may represent an emerging approach to discover therapeutic agents for gastric cancer, such as vorinostat. The observation of altered gene expression after vorinostat treatment may provide the clue to identify the molecular mechanism of vorinostat and those patients likely to benefit from vorinostat treatment.     

MicroRNA-30c serves as an independent biochemical recurrence predictor and potential tumor suppressor for prostate cancer

MicroRNA-30c (miR-30c) acts as a tumor suppressor or a tumor promoter in various human malignancies. However, the involvement of miR-30c in prostate cancer (PCa) is still unclear. The aim of this study was to investigate the molecular function and the clinical significance of miR-30c in PCa. Expression levels of miR-30c in PCa tissues and cells were detected by quantitative real-time-PCR (qRT-PCR). Additionally, the associations of miR-30c expression with clinicopathological features and prognosis in PCa patients were analyzed. The potential role of miR-30c in tumorigenesis of PCa cells was further evaluated by in vitro cell assays. MiR-30c was significantly down-regulated in PCa tissues and cells compared with the corresponding controls (P < 0.05). In addition, the downregulation of miR-30c in PCa tissues was significantly associated with higher Gleason score (P = 0.009), advanced pathological stage (P = 0.016) and biochemical recurrence (P = 0.034). Moreover, Kaplan–Meier survival analysis showed that the reduced expression of miR-30c was correlated with shorter biochemical recurrence-free survival (P = 0.023). The multivariate analysis also identified miR-30c as an independent prognostic predictor for biochemical recurrence-free survival in patients with PCa. Furthermore, the enforced expression of miR-30c suppressed proliferation, migration and invasion of PCa cells in vitro. Our data indicated the involvement of miR-30c in PCa progression and suggested its potential role as an independent predictor of biochemical recurrence in PCa. On cellular level, miR-30c may function as a tumor suppressor for PCa cells by inhibiting tumor cell proliferation, migration and invasion.     

Integrated analysis of multiple microarray datasets identifies a reproducible survival predictor in ovarian cancer

Background

Public data integration may help overcome challenges in clinical implementation of microarray profiles. We integrated several ovarian cancer datasets to identify a reproducible predictor of survival.

Methodology/Principal Findings

Four microarray datasets from different institutions comprising 265 advanced stage tumors were uniformly reprocessed into a single training dataset, also adjusting for inter-laboratory variation (“batch-effect”). Supervised principal component survival analysis was employed to identify prognostic models. Models were independently validated in a 61-patient cohort using a custom array genechip and a publicly available 229-array dataset. Molecular correspondence of high- and low-risk outcome groups between training and validation datasets was demonstrated using Subclass Mapping. Previously established molecular phenotypes in the 2^nd validation set were correlated with high and low-risk outcome groups. Functional representational and pathway analysis was used to explore gene networks associated with high and low risk phenotypes. A 19-gene model showed optimal performance in the training set (median OS 31 and 78 months, p<0.01), 1^st validation set (median OS 32 months versus not-yet-reached, p = 0.026) and 2^nd validation set (median OS 43 versus 61 months, p = 0.013) maintaining independent prognostic power in multivariate analysis. There was strong molecular correspondence of the respective high- and low-risk tumors between training and 1^st validation set. Low and high-risk tumors were enriched for favorable and unfavorable molecular subtypes and pathways, previously defined in the public 2^nd validation set.

Conclusions/Significance

Integration of previously generated cancer microarray datasets may lead to robust and widely applicable survival predictors. These predictors are not simply a compilation of prognostic genes but appear to track true molecular phenotypes of good- and poor-outcome.     

Gene-gene interaction analysis identifies a new genetic risk factor for colorectal cancer

Background

Adiponectin levels have been shown to be associated with colorectal cancer (CRC). Furthermore, a newly identified adiponectin receptor, T-cadherin, has been associated with plasma adiponectin levels. Therefore, we investigated the potential for a genetic association between T-cadherin and CRC risk.

Result

We conducted a case–control study using the Korean Cancer Prevention study-II cohort, which is composed of 325 CRC patients and 977 normal individuals. Study results revealed that rs3865188 in the 5’ flanking region of the T-cadherin gene (CDH13) was significantly associated with CRC (p = 0.0474). The odds ratio (OR) for the TT genotype as compared to the TA + AA genotype was 1.577 (p = 0.0144). In addition, the interaction between CDH13 and the adiponectin gene (APN) for CRC risk was investigated using a logistic regression analysis. Among six APN single nucleotide polymorphisms (rs182052, rs17366568, rs2241767, rs3821799, rs3774261, and rs6773957), an interaction with the rs3865188 was found for four (rs2241767, rs3821799, rs3774261, and rs6773957). The group with combined genotypes of TT for rs3865188 and GG for rs377426 displayed the highest risk for CRC development as compared to those with the other genotype combinations. The OR for the TT/GG genotype as compared to the AA/AA genotype was 4.108 (p = 0.004). Furthermore, the plasma adiponectin level showed a correlation with the gene-gene interaction, and the group with the highest risk for CRC had the lowest adiponectin level (median, 4.8 μg/mL for the TT/GG genotype vs.7.835 μg/mL for the AA/AA genotype, p = 0.0017).

Conclusions

The present study identified a new genetic factor for CRC risk and an interaction between CDH13 and APN in CRC risk. These genetic factors may be useful for predicting CRC risk.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0180-9) contains supplementary material, which is available to authorized users.Keyword: Gene-gene interaction, CRC, CDH13, rs3865188, APNSNPs     

Stem cell gene Girdin: a potential early liver metastasis predictor of colorectal cancer

The objective of this study is to investigate the liver's metastasis-related genes and the relationship between Girdin protein expression and clinical and pathological characteristics and prognosis in colorectal cancer. The differential expression of genes between tumor cells from cases with liver metastasis and those from cases without liver metastasis were detected using an RT(2) Profiler? PCR Array. The expression of the stem cell gene Girdin was analyzed using immunohistochemistry staining. Subsequently, the relationship between Girdin and clinicopathological parameters of colorectal cancer was determined. The Girdin protein was verified as a gene related to liver metastasis and was expressed positively in 161 (37.01 %) of the 435 cases examined. The expression of Girdin protein was related to histological grade and distant metastasis (P = 0.007 and 0.007, respectively). After analyzing survival rates, cases with highly expressed Girdin protein were shown to attain a significantly higher rate of liver metastasis and poorer postoperative, disease-specific survival than those with no or low expressed Girdin protein (P = 0.001). In the Cox regression test, the depth of tumor invasion, histological grade, duke's stage, distant metastasis, and the Girdin protein were detected as an independent prognostic factor (0.020, 0.032, 0.001, 0.001, and 0.010, respectively). The Girdin protein may be a potential new early liver metastasis biomarker of colorectal cancer.     

Epigenetic profiling and mRNA expression reveal candidate genes as biomarkers for colorectal cancer

Colorectal cancer (CRC) is characterized by DNA methylation, which is associated with genomic instability and tumor initiation. As an important epigenetic regulation, DNA methylation can be used as a potential therapeutic target for CRC. In our study, we downloaded DNA methylation profiles (GSE17648 and GSE29490) and RNA sequencing microarray data (GSE25070 and GSE32323) from the Gene Expression Omnibus (GEO) database. As a result, 14 aberrantly methylated differentially expressed genes (DEGs) were screened according to the different criteria. We further validated these DEGs in The Cancer Genome Atlas (TCGA) database and obtained Pearson's correlation coefficient (COR) for the relationship between gene expression and DNA methylation. Three candidate genes (SOX9, TCN1, and TGFBI) with COR greater than 0.3 were screened out as Hub genes. The receiver operating characteristic result indicated that SOX9 and TGFBI effectively serve as biomarkers for the early diagnosis of CRC. Furthermore, the potential prognosis of the Hub genes for CRC patients was evaluated. Only TGFBI, which is regulated by methylation, can predict patient disease-free survival. Additionally, we examined the methylation level of the Hub genes in CRC cells in the Cancer Cell Line Encyclopedia database. Considering that methylation status tends to be highly modified on CpG islands in tumorigenesis, we screened the CpG island methylation of TGFBI based on the TCGA database and verified its diagnostic value in the GEO database. Our result revealed two Hub genes (TCN1 and TGFBI) whose aberrant expressions were regulated by DNA methylation. Additionally, we uncovered the hypermethylation of TGFBI on CpG islands and its clinical value in the diagnosis of CRC.     

Genome-wide analysis identifies interleukin-10 mRNA as target of tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein required for the rapid degradation of mRNAs containing AU-rich elements. Targets regulated by TTP include the mRNAs encoding tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor, interleukin-2 (IL-2), and immediate early response 3. To identify novel target mRNAs of TTP in macrophages, we used a genome-wide approach that combines RNA immunoprecipitation and microarray analysis. A list was compiled of 137 mRNAs that are associated with TTP with an estimated accuracy on the order of 90%. Sequence analysis revealed a highly significant enrichment of AU-rich element motifs, with AUUUA pentamers present in 96% and UUAUUUAUU nonamers present in 44% of TTP-associated mRNAs. We further show that IL-10 is a novel target regulated by TTP. IL-10 mRNA levels were found to be elevated because of a reduced decay rate in primary macrophages from TTP(-/-) mice. Our study demonstrates the importance of experimental approaches for identifying targets of RNA-binding proteins.     

MAGE-C2/CT10 protein expression is an independent predictor of recurrence in prostate cancer

The cancer-testis (CT) family of antigens is expressed in a variety of malignant neoplasms. In most cases, no CT antigen is found in normal tissues, except in testis, making them ideal targets for cancer immunotherapy. A comprehensive analysis of CT antigen expression has not yet been reported in prostate cancer. MAGE-C2/CT-10 is a novel CT antigen. The objective of this study was to analyze extent and prognostic significance of MAGE-C2/CT10 protein expression in prostate cancer. 348 prostate carcinomas from consecutive radical prostatectomies, 29 castration-refractory prostate cancer, 46 metastases, and 45 benign hyperplasias were immunohistochemically analyzed for MAGE-C2/CT10 expression using tissue microarrays. Nuclear MAGE-C2/CT10 expression was identified in only 3.3% primary prostate carcinomas. MAGE-C2/CT10 protein expression was significantly more frequent in metastatic (16.3% positivity) and castration-resistant prostate cancer (17% positivity; p<0.001). Nuclear MAGE-C2/CT10 expression was identified as predictor of biochemical recurrence after radical prostatectomy (p = 0.015), which was independent of preoperative PSA, Gleason score, tumor stage, and surgical margin status in multivariate analysis (p<0.05). MAGE-C2/CT10 expression in prostate cancer correlates with the degree of malignancy and indicates a higher risk for biochemical recurrence after radical prostatectomy. Further, the results suggest MAGE-C2/CT10 as a potential target for adjuvant and palliative immunotherapy in patients with prostate cancer.     

ERp29 forms a feedback regulation loop with microRNA-135a-5p and promotes progression of colorectal cancer

Expression of endoplasmic reticulum (ER) stress-associated genes is often dysregulated in cancer progression. ER protein 29 (ERp29) is abnormally expressed in many neoplasms and plays an important role in tumorigenesis. Here, we showed ERp29 is a novel target for microRNA-135a-5p (miR-135a-5p) to inhibit the progression of colorectal cancer (CRC); correspondingly, ERp29 acts as an oncoprotein in CRC by promoting proliferation and metastasis of CRC cells, and suppressing apoptosis of the cells. More importantly, we found that miR-135a-5p expression is reversely upregulated by ERp29 through suppressing IL-1β-elicited methylation of miR-135a-5p promoter region, a process for enterocyte to maintain a balance between miR-135a-5p and ERp29 but dysregulated in CRC. Our study reveals a novel feedback regulation loop between miR-135a-5p and ERp29 that is critical for maintaining appropriate level of each of them, but partially imbalanced in CRC, resulting in abnormal expression of miR-135a-5p and ERp29, which further accelerates CRC progression. We provide supporting evidence for ERp29 and miR-135a-5p as potential biomarkers for diagnosis and treatment of CRC.Subject terms: Cell death, Oncogenes     

Wrist actimetry circadian rhythm as a robust predictor of colorectal cancer patients survival

The disruption of the circadian timing system (CTS), which rhythmically controls cellular metabolism and proliferation, accelerated experimental cancer progression. A measure of CTS function in cancer patients could thus provide novel prediction information for outcomes, and help to identify novel specific therapies. The rest-activity circadian rhythm is a reliable and non-invasive CTS biomarker, which was monitored using a wrist watch accelerometer for 2 days in 436 patients with metastatic colorectal cancer. The relative percentage of activity in-bed versus out-of-bed (I?<?O) constituted the tested CTS measure, whose prognostic value for overall survival (OS) and progression-free survival (PFS) was determined in a pooled analysis of three patient cohorts with different treatment exposures. Median OS was 21.6 months [17.8–25.5] for patients with I?<?O above the median value of 97.5% as compared to 11.9 months [10.4–13.3] for those with a lower I?<?O (Log-rank p?<?0.001). Multivariate analyses retained continuous I?<?O as a joint predictor of both OS and PFS, with respective hazard ratios (HR) of 0.954 (p?<?0.001) and 0.970 (p?<?0.001) for each 1% increase in I?<?O. HRs had similar values in all the patient subgroups tested. The circadian physiology biomarker I?<?O constitutes a robust and independent quantitative predictor of cancer patient outcomes, that can be easily and cost-effectively measured during daily living. Interventional studies involving 24-h schedules of clock-targeted drugs, light intensity, exercise and/or meals are needed for testing the relevance of circadian synchronization for the survival of patients with disrupted rhythms.     

Functional genetic screen identifies ITPR3/calcium/RELB axis as a driver of colorectal cancer metastatic liver colonization

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In silico analysis of angiogenesis associated gene expression identifies angiogenic stage related profiles

In vitro models have been extensively used to map gene expression in ECs but few studies have used cells from in vivo sources directly. Here, we compare different gene expression surveys on both cultured and fresh tissue derived ECs, and it emerges that gene expression profiles can be paralleled with the angiogenic stage of the cells. ECs stimulated with different growth factors in monolayer cultures exhibit gene expression profiles indicative of an active proliferative state, whereas gene expression in tube forming cells in vitro involves genes implicated in cell adhesion processes. Genes overexpressed in tumor ECs are biased towards extracellular matrix remodeling, a late event in angiogenesis. The elucidation of gene expression profiles under these different conditions will contribute to a better understanding of the molecular mechanisms during angiogenesis in both pathological and physiological circumstances and will have implications for the development of angiogenesis interfering treatment strategies.     

Functional expression cloning identifies COX-2 as a suppressor of antigen-specific cancer immunity

The efficacy of immune surveillance and antigen-specific cancer immunotherapy equally depends on the activation of a sustained immune response targeting cancer antigens and the susceptibility of cancer cells to immune effector mechanisms. Using functional expression cloning and T-cell receptor (TCR) transgenic mice, we have identified cyclooxygenase 2/prostaglandin-endoperoxide synthase 2 (COX-2) as resistance factor against the cytotoxicity induced by activated, antigen-specific T cells. Expressing COX-2, but not a catalytically inactive COX-2 mutant, increased the clonogenic survival of E1A-transformed murine cancer cells when cocultured with lymphocytes from St42Rag2^−/− mice harboring a transgenic TCR directed against an E1A epitope. COX-2 expressing tumors established in immune-deficient mice were less susceptible to adoptive immunotherapy with TCR transgenic lymphocytes in vivo. Also, immune surveillance of COX-2-positive tumor cells in TCR transgenic mice was less efficient. The growth of murine MC-GP tumors, which show high endogenous COX-2 expression, in immunocompetent mice was effectively suppressed by treatment with a selective COX-2 inhibitor, celecoxib. Mechanistically, COX-2 expression blunted the interferon-gamma release of antigen-specific T cells exposed to their respective cellular targets, and increased the expression of interleukin-4 and indoleamine 2,3-dioxygenase by tumor cells. Addition of interferon-gamma sensitized COX-2 expressing cancer cells to tumor suppression by antigen-specific T cells. In conclusion, COX-2, which is frequently induced in colorectal cancer, contributes to immune evasion and resistance to antigen-specific cancer immunotherapy by local suppression of T-cell effector functions.Anticancer immunity mediates immune surveillance and may be exploited for cancer immunotherapy. It involves innate immunity and natural killer cells, and antigen-specific immunity directed against cancer-specific antigens and viral antigens. Several escape mechanisms from cancer-specific immune surveillance and immunotherapy have been described. These comprise defective antigen processing and presentation by downmodulation of major histocompatibility complex (MHC) expression as well as immune editing of the antigen repertoire of a given cancer.¹ Upregulated inhibitory ligands, such as PD-L1, and secreted factors like indoleamine 2,3-dioxygenase (IDO, encoded by IDO1) functionally suppress antigen-presenting cells and cytotoxic cellular immune effectors.^{2, 3} In addition, cell-autonomous mechanisms may decrease susceptibility of cancer to immune effector mechanisms. These involve granule-dependent cytotoxicity involving perforin and granzymes, death receptor-induced apoptosis, complement-dependent cytotoxicity and secreted factors such as interferons, all of which trigger specific intracellular death pathways.^{4, 5, 6, 7, 8} Accordingly, the success of immune prevention and immunotherapy relies on both, the activation of a potent immune response against cancer and its susceptibility to immune elimination.Clinically applied modalities of cancer immunotherapy include the adoptive transfer of cellular immune effectors by means of allogeneic stem cell transplantation and donor lymphocyte therapy, monoclonal antibodies with direct and indirect cytotoxic mechanisms, and active immunotherapy with cellular and acellular vaccines.⁹ Moreover, immune regulatory interventions using cytokines and, more recently, immune regulatory antibodies directed against CTLA-4, PD-1 and PD-L1 have been employed with varying success.^{10, 11} A detailed understanding of the activation and regulation of a cancer-specific immune reaction as well as the determinants of efficacy of the effector phase of immune elimination is crucial for successful implementation and improvement of such immunotherapies. To this end we have developed experimental systems for unbiased identification of cell-autonomous mechanisms that modulate the susceptibility of cancer to the cytotoxic effects of activated, antigen-specific T cells. We identify cyclooxygenase 2/prostaglandin-endoperoxide synthase 2 (COX-2), a pathogen-induced enzyme involved in prostaglandin synthesis, as mediator of resistance to the effector phase of antigen-specific cancer immunity. Deregulation of COX-2 has been implied in the pathogenesis of several cancers, in particular colorectal cancer, where it impacts on oncogenic signaling, invasion and metastasis, survival and angiogenesis.^{12, 13, 14, 15} Moreover, COX-2-dependent prostaglandin release can suppress antigen presentation and immune activation in cancer.¹⁶ Here we describe COX-2 as a suppressor of antigen-induced interferon-gamma secretion of T cells and inducer of immunosuppressive factors that contributes to escape from immune surveillance and resistance to cellular immunotherapy. COX-2 may serve as predictive biomarker and as therapeutic target for modulation of immune resistance in cancer.     

Piceatannol promotes apoptosis via up-regulation of microRNA-129 expression in colorectal cancer cell lines

Piceatannol, a naturally occurring analog of resveratrol, has been confirmed as an antitumor agent by inhibiting proliferation, migration, and metastasis in diverse cancer. However, the effect and mechanisms of piceatannol on colorectal cancer (CRC) have not been well understood. This study aimed to test whether piceatannol could inhibit growth of CRC cells and reveal its underlying molecular mechanism.     

Multi-modality imaging identifies key times for annexin V imaging as an early predictor of therapeutic outcome

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.