A workflow for the enrichment, the identification, and the isolation of non-apoptotic single circulating tumor cells for RNA sequencing analysis.

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

DanioCTC: Analysis of Circulating Tumor Cells from Metastatic Breast Cancer Patients in Zebrafish Xenografts.

Circulating tumor cells (CTCs) serve as crucial metastatic precursor cells, but their study in animal models has been hindered by their low numbers. To address this challenge, we present DanioCTC, an innovative xenograft workflow that overcomes the scarcity of patient-derived CTCs in animal models. By combining diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the CellCelector setup, DanioCTC effectively enriches and isolates CTCs from metastatic breast cancer (MBC) patients for injection into zebrafish embryos. Validation experiments confirmed that MDA-MB-231 cells, transplanted following the standard protocol, localized frequently in the head and blood-forming regions of the zebrafish host. Notably, when MDA-MB-231 cells spiked (i.e., supplemented) into DLA aliquots were processed using DanioCTC, the cell dissemination patterns remained consistent. Successful xenografting of CTCs from a MBC patient revealed their primary localization in the head and trunk regions of zebrafish embryos. DanioCTC represents a major step forward in the endeavors to study the dissemination of individual and rare patient-derived CTCs, thereby enhancing our understanding of metastatic breast cancer biology and facilitating the development of targeted interventions in MBC. Summary statement: DanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies of the capacity of these rare tumor cells to induce metastases.

Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy.

Ablative radiotherapy is a highly efficient treatment modality for patients with metastatic prostate cancer (PCa). However, a subset of patients does not respond. Currently, this subgroup with bad prognosis cannot be identified before disease progression. We hypothesize that markers indicative of radioresistance, stemness and/or bone tropism may have a prognostic potential to identify patients profiting from metastases-directed radiotherapy. Therefore, circulating tumor cells (CTCs) were analyzed in patients with metastatic PCa (n = 24) during radiotherapy with CellSearch, multicolor flow cytometry and imaging cytometry. Analysis of copy-number alteration indicates a polyclonal CTC population that changes after radiotherapy. CTCs were found in 8 out of 24 patients (33.3%) and were associated with a shorter time to biochemical progression after radiotherapy. Whereas the total CTC count dropped after radiotherapy, a chemokine receptor CXCR4-expressing subpopulation representing 28.6% of the total CTC population remained stable up to 3 months. At once, we observed higher chemokine CCL2 plasma concentrations and proinflammatory monocytes. Additional functional analyses demonstrated key roles of CXCR4 and CCL2 for cellular radiosensitivity, tumorigenicity and stem-like potential in vitro and in vivo. Moreover, a high CXCR4 and CCL2 expression was found in bone metastasis biopsies of PCa patients. In summary, panCK+ CXCR4+ CTCs may have a prognostic potential in patients with metastatic PCa treated with metastasis-directed radiotherapy.

Comparative analysis of EpCAM high-expressing and low-expressing circulating tumour cells with regard to their clonal relationship and clinical value.

BACKGROUND: Circulating tumour cells (CTCs) are mainly enriched based on the epithelial cell adhesion molecule (EpCAM). Although it was shown that an EpCAM low-expressing CTC fraction is not captured by such approaches, knowledge about its prognostic and predictive relevance and its relation to EpCAM-positive CTCs is lacking. METHODS: We developed an immunomagnetic assay to enrich CTCs from metastatic breast cancer patients EpCAM independently using antibodies against Trop-2 and CD-49f and characterised their EpCAM expression. DNA of single EpCAM high expressing and low expressing CTCs was analyzed regarding chromosomal aberrations and predictive mutations. Additionally, we compared CTC-enrichment on the CellSearch system using this antibody mix and the EpCAM based enrichment. RESULTS: Both antibodies acted synergistically in capturing CTCs. Patients with EpCAM high-expressing CTCs had a worse overall and progression-free survival. EpCAM high- and low-expressing CTCs presented similar chromosomal aberrations and mutations indicating a close evolutionary relationship. A sequential enrichment of CTCs from the EpCAM-depleted fraction yielded a population of CTCs not captured EpCAM dependently but harbouring predictive information. CONCLUSIONS: Our data indicate that EpCAM low-expressing CTCs could be used as a valuable tumour surrogate material-although they may be prognostically less relevant than EpCAM high-expressing CTCs-and have particular benefit if no CTCs are detected using EpCAM-dependent technologies.

Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions.

Cell loss during detection and isolation of circulating tumor cells (CTCs) is a challenge especially when label-free pre-enrichment technologies are used without the aid of magnetic particles. Although microfluidic systems can remove the majority of "contaminating" white blood cells (WBCs), their remaining numbers are still impeding single CTC isolation, thus making additional separation steps needed. This study aimed to develop a workflow from blood-to-single CTC for complex cell suspensions by testing two microwell formats. In the first step, different cell lines were used to compare the performances of Sievewell™ 370 K (TOK, Japan) and CellCelector™ Nanowell U25 (ALS Automated Lab Solutions, Germany) slides for cell labelling and single-cell micromanipulation. Confounding levels of auto-fluorescence inherent to different plastic materials used to cast the microwells, staining recovery rates, and cell isolation rates were determined. In the second step, three different blood preservation tubes were tested for RNA analysis. Lastly, the established workflow was applied to isolate CTCs from peripheral blood samples obtained from metastasized breast cancer (mBC) patients for single-cell DNA and RNA analysis. The detection of CTCs in Sievewell slides profit from better signal-to-noise ratios in the fluorescence channels mainly used for CTC detection. In addition, due to its design, Sievewell supports direct in situ CTC labelling, which minimizes cell loss and leads to single-cell recovery rates after staining of approx. 94%. Detection of PIK3CA mutations in single CTCs verified the applicability of the workflow for the analysis of genomic DNA of CTCs. Furthermore, combined with blood preservation up to 48 h at room temperature in LBguard tubes, panel RT-PCR transcript analysis was successful for single cell line cells and CTCs, respectively. The combined use of Sievewell microwell slides and CellCelector™ automated micromanipulation system improves single CTC detection, labelling and isolation from complex cell suspensions. This approach is especially valuable when samples of high cellular content are processed.

2q35-rs13387042 variant and the risk of breast cancer: a case-control study.

BACKGROUND: Breast Cancer is the most frequent neoplasm diagnosed among women worldwide. Genetic background and lifestyle/environment play a significant role in the disease etiology. According to Genome-wide association studies, some single-nucleotide polymorphisms such as 2q35-rs13387042-(G/A) have been introduced to be associated with breast cancer risk and features. In this study, we aimed to evaluate the association between this variant and the risk of breast cancer in a cohort of Iranian women. METHODS: Demographics and clinical information were collected by interview and using patients' medical records, respectively. DNA was extracted from 506 blood samples, including 184 patients and 322 controls, and genotyping was performed using allele specific-PCR. SPSS v16 was used for statistical analysis. RESULT: Statistically significant association was observed between AA genotype and disease risk in all patients [padj = 0.048; ORadj = 2.13, 95% CI (1.01-4.50)] and also ER-positive breast cancers [padj = 0.015; ORadj = 2.12, 95% CI (1.16-3.88)]. There was no association between rs13387042 and histopathological characteristics of the disease. Furthermore, overall survival was not statistically associated with genotype and allelic models even after adjustment for stage and receptor status (p > 0.05). CONCLUSION: There is a statistically significant association between 2q35-rs13387042 and breast cancer risk. rs13387042-AA genotype might be a risk-conferring factor for breast cancer development in the Iranian population. However, further consideration is suggested to confirm its role in risk assessment and probable association with other genetic markers.

Multiparametric Circulating Tumor Cell Analysis to Select Targeted Therapies for Breast Cancer Patients.

BACKGROUND: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof-of-concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients. METHODS: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs. RESULTS: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient's CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging. CONCLUSION: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens.

PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling.

In previous studies, we reported that progesterone receptor membrane component 1 (PGRMC1) is implicated in progestin signaling and possibly associated with increased breast cancer risk upon combined hormone replacement therapy. To gain mechanistic insight, we searched for potential PGRMC1 interaction partners upon progestin treatment by co-immunoprecipitation and mass spectrometry. The interactions with the identified partners were further characterized with respect to PGRMC1 phosphorylation status and with emphasis on the crosstalk between PGRMC1 and estrogen receptor α (ERα). We report that PGRMC1 overexpression resulted in increased proliferation of hormone receptor positive breast cancer cell lines upon treatment with a subgroup of progestins including norethisterone and dydrogesterone that promote PGRMC1-phosphorylation on S181. The ERα modulators prohibitin-1 (PHB1) and prohibitin-2 (PHB2) interact with PGRMC1 in dependency on S181-phosphorylation upon treatment with the same progestins. Moreover, increased interaction between PGRMC1 and PHBs correlated with decreased binding of PHBs to ERα and subsequent ERα activation. Inhibition of either PGRMC1 or ERα abolished this effect. In summary, we provide strong evidence that activated PGRMC1 associates with PHBs, competitively removing them from ERα, which then can develop its transcriptional activities on target genes. This study emphasizes the role of PGRMC1 in a key breast cancer signaling pathway which may provide a new avenue to target hormone-dependent breast cancer.

GLS-driven glutamine catabolism contributes to prostate cancer radiosensitivity by regulating the redox state, stemness and ATG5-mediated autophagy.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Evaluating the Association between CCR5delta32 Polymorphism (rs333) and the Risk of Breast Cancer in a Cohort of Iranian Population.

BACKGROUND: CC chemokine receptor 5 (CCR5) is introduced as an immune response modulator. The activity of CCR5 influences breast tumour development in a p53-dependent manner. This study aimed to investigate the frequency of CCR5delta32 and its association with the risk of breast cancer in 1038 blood samples in North East of Iran. METHODS: In this case-control study, we genotyped 570 control samples and 468 breast cancer patients by a gel electrophoresis-based gap-polymerase chain reaction (gap-PCR) method Mashhad, Iran. The data were analyzed using the SPSS software. RESULTS: Of 570 controls included, 542 (95.09%) had CCR5delta32 wild/wild (W/W) genotype, 28 samples (4.91%) had CCR5delta32 wild/deletion (W/D) genotype and none of them were CCR5delta32 deletion/deletion (D/D) genotype (0%). While 428 samples of patients (91.45%) had CCR5delta32 W/W genotype, 40 samples (8.55%) had CCR5delta32 W/D and CCR5delta32 D/D homozygous was nil (0%) amongst cases. All samples were in the Hardy-Weinberg equilibrium (P>0.05). According to the allele frequency, D allele, as a risky allele, in the cases was more than the control samples (0.0427 vs 0.0245, respectively) (P=0.0206). Hence, W/D genotype may confer a risk effect (OR=1.77, CI: 1.09-2.90; P=0.0206) compared with WW genotype between case and control groups. CONCLUSION: There is a statistically significant association between CCR5W/D and breast cancer risk. CCR5 may be regarded as a target for the prevention of breast cancer in certain conditions such as interaction with p53 variants, which remains to be further investigated.

Bioinformatic Identification of a Breast-Specific Transcript Profile.

PURPOSE: To identify a breast-specific transcript profile for the first time, and present an updated bioinformatics strategy for searching tissue-specific transcripts and predicting their significance in cancer. EXPERIMENTAL DESIGN: The RNA-seq data of 49 311 transcripts in 88 human tissues from the GTEx, the Illumina Body Map, and the RIKEN FANTOM5 project are integrated to screen breast-specific transcripts. Gene Expression Profiling Interactive Analysis, TGCA, and Kaplan-Meier Plotter are used to examine their expression in cancer tissues and values for prognosis prediction. RESULTS: Only 96 transcripts in human genome are breast-specific for women. Among them, ankyrin repeat domain 30A (ANKRD30A) and long intergenic non-protein coding RNA 993 (LINC00993) are further analyzed. The two transcripts are also breast-specific in 33 types of common female cancer and are often dysregulated in breast cancer tissues. Their expression is higher in the luminal breast cancer while significantly downregulated in triple-negative breast cancer. Moreover, the high expression levels of ANKRD30A and LINC0993 in breast cancer tissues indicate a better prognosis of patients with breast cancer. CONCLUSIONS AND CLINICAL RELEVANCE: Breast-specific transcripts in human genome are rare and poorly understood currently. The data indicate that these breast-specific biomarkers are promising candidates for screening early cancer, assessing treatment response, monitoring recurrence, identifying metastatic tumor origin, and serving as potential targets for immunotherapy.

The Dilemma of TP53 Codon 72 Polymorphism (rs1042522) and Breast Cancer Risk: A Case-Control Study and Meta-Analysis in The Iranian Population.

OBJECTIVE: Mutations of TP53 as a tumor suppressor gene are frequently observed in different types of cancer. A codon 72 polymorphism located on exon 4 with two alleles encoding either Proline (CCC) or Arginine (CGC) has been indicated as a common variation in association with cancers. Controversial results have been reported regarding the association of allelic polymorphism of codon 72 of TP53 gene and breast cancer risk in Iranian patients. Therefore, a case-control study was designed. A meta-analysis was also carried out to provide evidence of association between this variation and breast cancer in Iran, based on all available published data. MATERIALS AND METHODS: In this case-control study, blood sample of 622 participants, including 308 breast cancer cases and 314 controls were collected. Genotyping for rs1042522 was conducted by Allele Specific polymerase chain reaction (AS-PCR). In order to set a meta-analysis study, PubMed, Scopus and ISI Web of Knowledge and Persian databases were searched to explore relevant studies, published up to September 2018, containing information on TP53 polymorphism and the risk of breast cancer in Iran. Statistical analysis was performed using SPSS 16.0 and MetaGenyo. RESULTS: All retrieved available data as well as the results of our current study were consisted of 1965 breast cancer cases and 1999 healthy controls. No significant difference was observed in allele frequencies between groups (P=0.90) in our study. The cumulative results did not also show any association between rs1042522 and breast cancer risk on the dominant (P=0.61) and recessive (P=0.89) models. CONCLUSION: These findings cannot support contribution of rs1042522 polymorphism to breast cancer risk in an Iranian population. Future larger studies may help confirm this finding with a greater power.

Association of caspase 8 promoter variants and haplotypes with the risk of breast cancer and its molecular profile in an Iranian population: A case-control study.

Caspase 8 (CASP8) gene plays a key role in the regulation of apoptotic cell death. Expression variation in this gene has been associated with the risk of breast cancer. The aim of this study was to investigate the association of rs3834129 and rs3769821, as functional variants, and their haplotypes with molecular profile as well as the risk of breast cancer in an Iranian population. A case-control study was conducted on 812 participants including 293 breast cancer patients and 519 healthy controls. Genotyping was performed by polymerase chain reaction-based methods. Statistical analysis was performed using SPSS Ver16. The association between polymorphisms and haplotypes with the risk of breast cancer was estimated by calculating odds ratios (OR) and chi-square (χ2 ) tests. In comparison with ins allele (I) of rs3834129, carriers of del allele (D) showed a lower risk of breast cancer (OR, 0.65; 95% confidence interval [CI], 0.49-0.87; P = 0.004). The multivariate logistic regression model indicated DD genotype as an independent factor for a decreased risk of breast cancer in our population (OR, 0.18; 95% CI, 0.06-0.58; P = 0.004). Also, the C allele of rs3769821 was associated with a 43% increased risk of breast cancer (P = 0.005); however, after adjustment for confounding factors, no association with rs3769821 and breast cancer was observed. In addition, D-T haplotype and diplotype presented protective effects (P < 0.05). Our results indicate that genetic variations in the promoter region of CASP8 gene, especially rs3834129, may serve as a genetic risk factor for breast cancer in an Iranian population.

Regulators and mechanisms of anoikis in triple-negative breast cancer (TNBC): A review.

Metastasis leads to poor prognosis and reduced disease-free survival in breast cancer patients, particularly in those with triple-negative breast cancer (TNBC) which is resistant to common treatments. Anoikis is a type of apoptosis commenced by the detachment of cells from the native extracellular matrix and prohibits the attachment of detached cells to other body organs. Resistance to anoikis is a critical culprit in the development and progression of tumours. It is therefore important to understand the anoikis-related molecular pathways in order to design effective therapies for TNBC. Several compounds have been shown to possess the potential to regulate anoikis in breast cancer cells such as DSF, AEB071, nanoencapsulated doxorubicin, berberine, salinomycin, PEM POL5551, AL10, 5-azacytidine, synthesized flavonoid derivative GL-V9, Tubeimoside V (TBMS-V) and HPW-RX40. We reviewed the molecular basis of anoikis regulation, its potential role as an important target to inhibit metastasis in TNBC, and potential anoikis modulators that could serve as drug candidates.

The prognostic and therapeutic values of long noncoding RNA PANDAR in colorectal cancer.

Long noncoding RNAs (lncRNAs) consist of 200 nucleotide sequences that play essential roles in different processes, including cell proliferation, and differentiation. There is evidence showing that the dysregulation of lncRNAs promoter of CDKN1A antisense DNA damage-activated RNA (PANDAR) leads to the development and progression in several cancers including colorectal cancer, via p53-dependent manner. This suggests that these lncRNAs may be of value as prognostic indices and a therapeutic target, as a high expression of lncRNAs PANDAR is associated with poor prognosis. Furthermore, modulating lncRNAs PANDAR has been reported to induce apoptosis and inhibit the tumor growth through modulation of cell cycle and epithelial-mesenchymal transition (EMT) pathway. The aim of the current review was to provide an overview of the prognostic and therapeutic values of lncRNAs PANDAR in colorectal cancer.

Significant association of TOX3/LOC643714 locus-rs3803662 and breast cancer risk in a cohort of Iranian population.

Genome-wide association studies normally focus on low penetrance and moderate to high-frequency single nucleotide polymorphisms (SNPs), which lead to genetic susceptibility to breast cancer. In this regard, the T allele of rs3803662 has been associated with breast cancer risk and with lower expression level of TOX3. We aimed to assess the risk of breast cancer associated with this polymorphism in an Iranian population. Using Tetra Primer ARMS PCR, rs3803662 was analyzed in a total of 943 individuals (430 cases and 513 healthy controls form North East of Iran). Allele frequencies and genotype distribution were analyzed in case and control samples to find out any association using the Chi-squared test and Logistic regression. All cases were pathologically confirmed; all controls were mainly healthy individuals. Genotype frequencies were found to be in agreement with HWE in controls and cases. TOX3-rs3803662 SNP was associated with breast cancer risk in our study (T vs. C allele contrast model: OR 1.36, 95% CI 1.12-1.64, Pvalue = 0.002; TT vs. CT + TT dominant model: OR 0.67, 95% CI 0.51-0.87, Pvalue = 0.003; TT vs. CT + CC recessive model: OR 1.54, 95% CI 1.02-2.30, Pvlue = 0.036). Moreover, after adjusting for age, BMI, history of previous cancer and also family history of cancer, all results, except for the recessive model, were remained significant. TOX3-rs3803662, may confer some degrees of risk of breast cancer in Iranian population. This finding is in line with similar results in other populations. It highlights the importance of TOX3 pathway in tumorigenesis.

Evaluation of leishmanicidal effect of Euphorbia petiolata extract by in vivo anti-leishmanial assay using promastigotes of Leishmania major.

OBJECTIVE: The extract of different species of Euphorbia has been successfully used as a remedy for treatment of cutaneous leishmaniasis. The aim of this study was to assess the in vitro leishmanicidal effect of Euphorbia petiolata (E. petiolata) extract. MATERIALS AND METHODS: Ethanolic percolated and methanolic Soxhlet extract of E. petiolata on promastigotes of L. major at different concentrations of extracts, one positive control group and one negative control group as well as 1 solvent control were prepared and placed in 24-well plates that contained 40,000 parasites/well. Afterwards, plates were incubated at 25 ˚C for six days and number of parasites in each well were determined on days 2, 4 and 6 of the experiment. RESULTS: Both percolated and Soxhlet extracts in methanol and DMSO solvents had significant effects (equal to that of amphotericin B) on promastigote form of parasite at the concentration of 1 mg/ml. At lower concentrations, the extracts of E. petiolata had favorable leishmanicidal activity and killed L. major promastigotes dose-dependently. CONCLUSION: Our results support the possibility of E. petiolata extracts application as an anti-leishmanial agent with similar effects to amphotericin B.

Polymorphisms in non-coding RNAs and risk of colorectal cancer: A systematic review and meta-analysis.

Colorectal cancer (CRC) has been regarded as a common cancer due to its prevailing incidence in both males and females. Recently, non-coding RNAs used as biomarkers for screening, diagnosis and prognosis of different cancers have been under the focus of attention. As a result of this, the aim of this study was to systematically review articles that investigated the SNPs in genes related to microRNAs and long non-coding RNAs to assess the genetic susceptibility of colorectal cancer risk. The outcome is presented as the results of a meta-analysis. We systematically searched PubMed, Web of Science, and Scopus to identify relevant studies published up to 20/5/2017. These included eligible studies consisting of 23,581 patients and 22,697 controls. The conferred risk was estimated and presented using odds ratios (ORs) and 95% confidence intervals (CI). The Hardy-Weinberg equilibrium (HWE) was assessed by the goodness-of-fit chi-square test in all studies. The power of each study was also calculated based on the available results. Out of 27 different microRNAs which had published results, although most of the studies were under powered, miR-146a and miR-196a were amongst the most studied microRNAs. For five miRNAs (miR-196a, miR-146a, miR-27a, miR-499 and miR-149) which we performed a meta-analysis, miR-27a and miR-149 gene polymorphisms were associated with susceptibility to CRC. Other miRNAs did not show any effect on the CRC risk. Overall, significant association between miR-149 rs2292832 and susceptibility to cancer was identified in a recessive genetic model, TT/ (TC + CC) (OR = 1.19, 95% CI = 1.02-1.39, P = 0.02). On the other hand, rs895819 (miR-27a) GG carriers were more susceptible to CRC (OR = 1.47, 95% CI = 1.21-1.78, P = <0.05) in a recessive genetic model. Analysis of the data based on race revealed that rs2910164 (miR-146a) polymorphism may decrease the risk of CRC among Europeans, in a co dominant model [OR = 0.81, 95% CI 0.66-0.99, p = 0.04], but not among Asians. In conclusion, certain miRNAs (miR-27a and miR-149) may affect the CRC risk and can be regarded as genetic markers amongst different populations. LncRNAs still have to be studied more to reach a conclusion for their association with CRC risk.

Elucidating the Underlying Functional Mechanisms of Breast Cancer Susceptibility Through Post-GWAS Analyses.

Genome-wide association studies (GWAS) have identified more than 170 single nucleotide polymorphisms (SNPs) associated with the susceptibility to breast cancer. Together, these SNPs explain 18% of the familial relative risk, which is estimated to be nearly half of the total familial breast cancer risk that is collectively explained by low-risk susceptibility alleles. An important aspect of this success has been the access to large sample sizes through collaborative efforts within the Breast Cancer Association Consortium (BCAC), but also collaborations between cancer association consortia. Despite these achievements, however, understanding of each variant's underlying mechanism and how these SNPs predispose women to breast cancer remains limited and represents a major challenge in the field, particularly since the vast majority of the GWAS-identified SNPs are located in non-coding regions of the genome and are merely tags for the causal variants. In recent years, fine-scale mapping studies followed by functional evaluation of putative causal variants have begun to elucidate the biological function of several GWAS-identified variants. In this review, we discuss the findings and lessons learned from these post-GWAS analyses of 22 risk loci. Identifying the true causal variants underlying breast cancer susceptibility and their function not only provides better estimates of the explained familial relative risk thereby improving polygenetic risk scores (PRSs), it also increases our understanding of the biological mechanisms responsible for causing susceptibility to breast cancer. This will facilitate the identification of further breast cancer risk alleles and the development of preventive medicine for those women at increased risk for developing the disease.

The 9p21 locus: A potential therapeutic target and prognostic marker in breast cancer.

Breast cancer is an important cause of cancer related mortality in women. Despite extensive efforts to identify valid biomarkers for risk stratification, there are relatively few with proven clinical utility. It is recognized that genetic factors play a major role in determining susceptibility to breast cancer. Recent genome-wide-association-studies and gene expression analysis have demonstrated that a locus on chromosome 9p21, which contains three genes; CDKN2B (encoding p15ink4b), CDKN2A (encoding p16ink4a and p14ARF) and the 3' end of CDKN2BAS (an antisense noncoding RNA in the INK4 locus [ANRIL]) are associated with an increased risk of this malignancy. ANRIL has a post transcriptional modulatory activity, which has been shown to perturb the expression of nearby genes and may play an important role in coordinating tissue remodeling through regulation of cell proliferation, apoptosis, aging, extra-cellular matrix remodeling, and inflammatory response. However, the role of ANRIL is not well understood in breast cancer. Hypermethylation of the p14ARF and p16INK4a genes is found in some tumor types. Nevertheless, further studies are necessary to confirm the clinical utility of these putative markers in risk stratification, or assessing prognosis. In this review, we have summarized the prognostic and therapeutic potential of the p14ARF and p16INK4a genes in patients with breast cancer.