Tomasz Kolenda

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.

lncRNA in HNSCC: challenges and potential

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cause of cancer mortality in the world. Some progress has been made in the therapy of HNSCC, however treatment remains unsatisfactory. Recent studies have shown that different types of long non-coding RNAs (lncRNAs) are dysregulated in HNSCC and correlate with tumor progression, lymph node metastasis, clinical stage and poor prognosis. lncRNAs are a class of functional RNA molecules that can not be translated into proteins but can modulate the activity of transcription factors or regulate changes in chromatin structure. The lncRNAs might have potential of biomarker in HNSCC diagnosis, prognosis, prediction and targeted treatment. In this review we describe the potential role of lncRNAs as new biomarkers and discuss their features including source of origin, extraction methods, stability, detection methods and data normalization and potential function as biomarkers in HNSCC.

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.

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.

miRNAs Set Expression Profiles in Whole Blood During Prostate Cancer Patients Treatment

Background: Changes in expression profiles of the 5 selected miRNAs were analysed in a group of PC patients before treatment, after hormonotherapy and radiotherapy. Objective: Whether the expression profiles of the miRNAs may be useful for monitoring prostate cancer treatment. Methods: The initial study was carried out on 44 advanced prostate cancer patients and 41 healthy volunteers. The target group consisted of 39 PC patients. Blood for miRNA analysis was taken before treatment, after hormonotherapy and radiotherapy. The miRNAs were analysed by real-time PCR, followed by statistical analysis. Results: For the target group, the statistically significant differences in the expression level were found after radiotherapy: for miR-21 only in the group of patients above the cut-off value designed in the preliminary study (p=0.0369) and miR-100 for the whole group (p=0.0413) and for the above cut-off value group (p=0.0140). The differences between the levels of each miRNA between the high and low expression groups were statistically significant. The designed groups were stable during treatment. Inclusion to the high and low expression group levels did not influence the treatment result. Conclusion: The miRNAs studied in this work could not serve as biomarkers for the effectiveness of therapy for prostate cancer patients.

Immunotherapy in Patients with Recurrent and Metastatic Squamous Cell Carcinoma of the Head and Neck.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant cancer occurring in the head and neck area, approximately 90% of the cases. Even in the cases of primary radical treatment (surgical, concomitant chemoradiotherapy or radiotherapy alone), local recurrence or distal metastases are often observed. In patients with recurrent disease who are unable to receive radical treatment, the results of palliative chemotherapy are not satisfactory. The breakthrough in the therapy of advanced HNSCC was the approval of cetuximab in combination with chemotherapy in 2008. However, for almost a decade we have seen many negative studies with new treatment approaches including various types of molecules targeting epidermal growth factor receptors or vascular endothelial growth factor receptors, as well as different cytokines or cancer vaccines. Recently two new agents have been approved in the treatment of recurrent or metastatic HNSCC. These are immune-checkpoint inhibitors targeting PD1 (nivolumab and pembrolizumab) expressed mainly on T-cells. However, the results remain unsatisfactory - most of the patients do not respond to the treatment and some of the responding patients develop further progression. Many phase 3 studies are currently ongoing evaluating the efficacy of combinational treatment - anti-CTLA4 with anti-PD1 or anti-PD-L1. Very encouraging results have been shown in early phase studies evaluating the combination of immune-checkpoint inhibitors with tumor microenvironment immunosuppressive inhibitors. Undoubtedly, further research in the field of biomarkers for effective immunotherapy is needed in order to select a group of patients whose will benefit from this therapy, as the treatment is still ineffective in most patients.

Oncogenic BRAF mutations and p16 expression in melanocytic nevi and melanoma in the Polish population

Introduction Twenty-five – fifty percent of skin melanomas arise from nevi. Melanocyte proliferation is activated by BRAF V600E, then is arrested, but single nevi transform to melanomas. p16 controls arrest, and p16 loss may promote transformation. Aim To analyze BRAF V600E, p16 expression and melanocyte proliferation in dermal, compound and dysplastic nevi, cells of primary and metastatic melanoma in the Polish population. Material and methods One hundred and thirty-two nevi (dermal, compound, dysplastic) and 41 melanomas (in situ, primary, metastatic) were studied. BRAF was assessed by cobas® 4800 BRAFV600 Mutation Test, High Resolution Melting Assay validated with: pyrosequencing and immunohistochemistry. p16 and Ki67 expression was analyzed by IHC. Results Eighty-two percent of nevi and 57% of melanomas display BRAF V600E expression. Most dermal and compound nevi had > 50% of p16(+) cells. BRAF V600E dysplastic nevi had a low number of p16(+) cells. Nevi without BRAF V600E (WT), had 90% of cells p16(+). In 60% of in situ and primary melanomas, there was a low number of cells of p16(+). Fifty percent of WT metastatic melanoma and 33% of BRAF V600E showed a high level of p16. The number of Ki67(+) cells in dysplastic nevi was very low. In 25% of BRAF V600E melanomas in situ and 55% of WT, > 10% cells were Ki67(+). All BRAF V600E primary melanomas and 66% of WT had > 10% Ki67(+) cells. Twenty percent of BRAF V600E and WT metastases had > 10% of Ki67(+), however, 62% of BRAF V600E and 32% of WT samples had > 50% of Ki67(+) cells. Conclusions BRAFV600E and p16 are more frequent in nevi than in melanoma in vivo. A significantly higher p16 expression was observed in mutated nevi than in WT, while in melanoma it was just the opposite. The proliferation rate of melanoma cells negatively correlated with p16 expression.