Correction: Whole exome sequencing of high-risk neuroblastoma identifies novel non-synonymous variants.

[This corrects the article DOI: 10.1371/journal.pone.0273280.].

Whole exome sequencing of high-risk neuroblastoma identifies novel non-synonymous variants.

Neuroblastoma (NBL), one of the main death-causing cancers in children, is known for its remarkable genetic heterogeneity and varied patient outcome spanning from spontaneous regression to widespread disease. Specific copy number variations and single gene rearrangements have been proven to be associated with biological behavior and prognosis; however, there is still an unmet need to enlarge the existing armamentarium of prognostic and therapeutic targets. We performed whole exome sequencing (WES) of samples from 18 primary tumors and six relapse samples originating from 18 NBL patients. Our cohort consists of 16 high-risk, one intermediate, and one very low risk patient. The obtained results confirmed known mutational hotspots in ALK and revealed other non-synonymous variants of NBL-related genes (TP53, DMD, ROS, LMO3, PRUNE2, ERBB3, and PHOX2B) and of genes cardinal for other cancers (KRAS, PIK3CA, and FLT3). Beyond, GOSeq analysis determined genes involved in biological adhesion, neurological cell-cell adhesion, JNK cascade, and immune response of cell surface signaling pathways. We were able to identify novel coding variants present in more than one patient in nine biologically relevant genes for NBL, including TMEM14B, TTN, FLG, RHBG, SHROOM3, UTRN, HLA-DRB1, OR6C68, and XIRP2. Our results may provide novel information about genes and signaling pathways relevant for the pathogenesis and clinical course in high-risk NBL.

2D and 3D cell cultures - a comparison of different types of cancer cell cultures.

Cell culture is a widely used in vitro tool for improving our understanding of cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production and the development of tissue engineering. Most research regarding cancer biology is based on experiments using two-dimensional (2D) cell cultures in vitro. However, 2D cultures have many limitations, such as the disturbance of interactions between the cellular and extracellular environments, changes in cell morphology, polarity, and method of division. These disadvantages led to the creation of models which are more closely able to mimic conditions in vivo. One such method is three-dimensional culture (3D). Optimisation of the culture conditions may allow for a better understanding of cancer biology and facilitate the study of biomarkers and targeting therapies. In this review, we compare 2D and 3D cultures in vitro as well as different versions of 3D cultures.

Tumor microenvironment - Unknown niche with powerful therapeutic potential.

Head and neck squamous cell carcinomas (HNSCC) are in a group of cancers that are the most resistant to treatment. The survival rate of HNSCC patients has been still very low since last 20 years. The existence of relationship between oncogenic and surrounding cells is probably the reason for a poor response to treatment. Fibroblasts are an important element of tumor stroma which increases tumor cells ability to proliferate. Another highly resistance, tumorigenic and metastatic cell population in tumor microenvironment are cancer initiating cells (CICs). The population of cancer initiating cells can be found regardless of differentiation status of cancer and they seem to be crucial for HNSCC development. In this review, we describe the current state of knowledge about HNSCC biological and physiological tumor microenvironment.

Different levels of let-7d expression modulate response of FaDu cells to irradiation and chemotherapeutics.

The implication of the let-7 family in cancer development is multifaceted. The family acts as tumor suppressor miRNA although overexpression of let-7 has also been described in many types of cancer, including head and neck squamous cell carcinoma (HNSCC). The aim of this study includes whether different expression levels of let-7d has an influence on chemo- and radiosensitivity. FaDu cell line models with a gradually increased level of let-7d (models from A to E) were generated with the lentiviral system. Expression levels of pluripotency, chemo-radioresistance/apoptosis, and targets of mRNAs were analyzed by real-time reverse transcription-PCR (qRT-PCR). Radiosensitivity was analyzed using a clonogenic assay after irradiation. Response to cisplatin, 5-FU, doxorubicin, and paclitaxel was done with MTT assay. Statistically significant decrease of K-RAS (p = 0.0369) and CASPASE3 (p = 0.0342) were observed with the growing expression level of let-7d. Cisplatin, 5-FU and doxorubicin caused similar decreased of cell survival with the increase of let-7d level (p = 0.004, post-trend p = 0.046; p = 0.004, post trend p = 0.0005 and p<0.0001, post trend p = 0.0001, respectively). All models were resistant to paclitaxel, irrespective of let-7d expression levels. Only two of the generated models (A and C) were radiosensitive (p = 0.0002). CONCLUSION: the above results indicated that the level of let-7d expression is an important factor for cell response to irradiation and chemotherapeutics.

Unpredictable changes of selected miRNA in expression profile of HNSCC.

BACKGROUND: The necessity of prediction and treatment outcome improvement of HNSCC needs to find new biomarkers. miRNAs seem to be good candidate for that. OBJECTIVE: Analysis of selected 5 miRNAs (let-7d, miR-18a, miR-21, miR-205 and miR-375) as potential biomarkers that allows to distinguish tumor and healthy tissue taken from HNSCC patients. METHODS: Tumor and normal epithelial tissues were obtained from 75 HNSCC patients to analyze selected miRNAs. RESULTS: Analysis indicated significant increase of miR-21 and miR-205 in tumor when compared with healthy tissue (p= 0.0069 and p= 0.0029, respectively). There was a significant correlation between let-7d and miR-18a. let-7d was down-regulated in 34.67% cases, miR-18a in 29.33%, miR-21 in 20%, miR-205 in 30.67% and miR-375 in 52% cases. At the same time over-expression of let-7d was detected in 18.67% cases, miR-18a in 22.67%, miR-21 in 48%, miR-205 in 41.33% and miR-375 in 52% cases. There was no correlation between miRNA expression and clinical data and the course of illness. CONCLUSION: Our study indicated that miR-21 and miR-205 can be used to analyze the clarity of surgical margins and that concomitant changes in the expression of let-7 and miR-18a in tumor tissues might represent important future markers indicating the biology of HNSCC. These observations will help with developing personalization for HNSCC patients' treatment.

The mystery of let-7d - a small RNA with great power.

miRNAs belong to a class of small non-coding RNAs which can modulate gene expression. Disturbances in their expression and function may cause cancer formation, progression and cell response to various types of stress. The let-7 family is one of the most studied groups of miRNAs. The family contains 13 members with similar sequences and a wide spectrum of target genes. In this paper, we mostly focus on one member of the family - let-7d. This miRNA is dysregulated in many types of cancers. It can be over- or down-expressed, and it acts as a tumor suppressor or oncogene. It regulates various genes such as LIN28, C-MYC, K-RAS, HMGA2 and IMP-1. Moreover, let-7d has a significant impact on epithelial-to-mesenchymal transition (EMT) and formation of cancer initiating cells which are resistant to irradiation and chemical exposure and responsible for cancer metastasis. Let-7d can serve as a prognostic and predictive marker for personalization of the treatment. Let-7d is a small RNA with great power, but in different cell genetic backgrounds it acts in different ways, which makes this molecule still mysterious.