Targeting Cell Death Mechanism Specifically in Triple Negative Breast Cancer Cell Lines.

Triple negative breast cancer (TNBC) is currently associated with a lack of treatment options. Arsenic derivatives have shown antitumoral activity both in vitro and in vivo; however, their mode of action is not completely understood. In this work we evaluate the response to arsenate of the double positive MCF-7 breast cancer cell line as well as of two different TNBC cell lines, Hs578T and MDA-MB-231. Multimodal experiments were conducted to this end, using functional assays and microarrays. Arsenate was found to induce cytoskeletal alteration, autophagy and apoptosis in TNBC cells, and moderate effects in MCF-7 cells. Gene expression analysis showed that the TNBC cell lines' response to arsenate was more prominent in the G2M checkpoint, autophagy and apoptosis compared to the Human Mammary Epithelial Cells (HMEC) and MCF-7 cell lines. We confirmed the downregulation of anti-apoptotic genes (MCL1, BCL2, TGFß1 and CCND1) by qRT-PCR, and on the protein level, for TGFß2, by ELISA. Insight into the mode of action of arsenate in TNBC cell lines it is provided, and we concluded that TNBC and non-TNBC cell lines reacted differently to arsenate treatment in this particular experimental setup. We suggest the future research of arsenate as a treatment strategy against TNBC.

Hsa-miR-125b Therapeutic Role in Colon Cancer Is Dependent on the Mutation Status of the TP53 Gene.

Colon cancer is the third most common cancer type worldwide and is highly dependent on DNA mutations that progressively appear and accumulate in the normal colon epithelium. Mutations in the TP53 gene appear in approximately half of these patients and have significant implications in disease progression and response to therapy. miR-125b-5p is a controversial microRNA with a dual role in cancer that has been reported to target specifically TP53 in colon adenocarcinomas. Our study investigated the differential therapeutic effect of miR-125b-5p replacement in colon cancer based on the TP53 mutation status of colon cancer cell lines. In TP53 mutated models, miR-125b-5p overexpression slows cancer cells' malignant behavior by inhibiting the invasion/migration and colony formation capacity via direct downregulation of mutated TP53. In TP53 wild type cells, the exogenous modulation of miR-125b-5p did not significantly affect the molecular and phenotypic profile. In conclusion, our data show that miR-125b-5p has an anti-cancer effect only in TP53 mutated colon cancer cells, explaining partially the dual behavior of this microRNA in malignant pathologies.

The Clinical Utility of miR-21 and let-7 in Non-small Cell Lung Cancer (NSCLC). A Systematic Review and Meta-Analysis.

Non-small cell lung cancer (NSCLC) remains a problem worldwide due to its rapid progression and low rate of response to treatment. The heterogeneity of these tumors observed in histopathology exam but also in the mutational status and gene expression pattern makes this malignancy difficult to treat in clinic. The present study investigated the effect of miR-21 and let-7 family members as prognostic biomarkers in NSCLC patients based on the results published in different studies regarding this subject until March 2019. The analysis revealed that these two transcripts are steady biomarkers for prediction of patient outcome or survival. Upregulated expression of miR-21 is associated with poor outcome of patients with NSCLC [HR = 1.87, 95% CI = (1.41, 2.47), p < 0.001]. The analysis regarding let-7 family, specifically let-7a/b/e/f, revealed that downregulated expression of these transcripts predicts poor outcome for NSCLC patients [HR = 2.61, 95% CI = (1.58, 4.30), p < 0.001]. Besides, the reliability of these microRNAs is reflected in the fact that their prognostic significance is constant given the different sample types (tissue, FFPE tissue, serum, serum/plasma or exosomes) used in the selected studies.

Circulating microRNA-194 and microRNA-1228 Could Predict Colon Cancer Proliferation via Phospho S6 Modulation.

BACKGROUND AND AIMS: Although colon cancer has a decreasing incidence trend in Europe, because of its still high frequency and not fully understood pathogenesis, this malignancy still remains a subject of intense research. The aim of this study was to investigate the role of microRNA-194 and microRNA-1228 in colon cancer proliferation. METHODS: RNA was extracted from patients with colon cancer with or without advanced disease and microRNA expression levels were determined through qRT-PCR. Assays were performed on HCT116 cell line and included qRT-PCR, western blotting and cell counting. RESULTS: We observed that both microRNAs 194 and 1228 were altered in patients with colon cancer compared with healthy individuals. We observed a lower expression of both microRNA-194 and microRNA-1228 in patients with advanced colon cancer. To validate their pathogenetic role we performed viability and invasion assays on HCT116 cell line transfected with mimics or inhibitors of the mentioned microRNAs, with observable changes in viability and invasion. Furthermore, to determine the altered signaling induced by these microRNAs, we performed western blotting for phospho S6 on HCT116 cells transfected with mimic and inhibitor of the above-mentioned microRNAs with observable differences. CONCLUSION: In the current study we have shown that both microRNA-194 and microRNA-1228 alteration was correlated with the presence of advanced colon cancer, a fact that was further validated in vitro through an invasion assay. Moreover, we have also shown that their effect might be mediated through phospho S6 expression.

Plasma and Tissue Specific miRNA Expression Pattern and Functional Analysis Associated to Colorectal Cancer Patients.

An increasing number of studies suggest the implication of microRNAs (miRNAs) in colorectal (CRC) carcinogenesis and disease progression. Nevertheless, the basic mechanism is not yet clear. We determined plasma miRNA expression levels using Agilent microarray technology followed by overlapping with The Cancer Genome Atlas (TCGA) tissue data and a qRT-PCR validation step and analysis of the altered miRNA signatures to emphasize new mechanistic insights. For TGCA dataset, we identified 156 altered miRNAs (79 downregulated and 77 upregulated) in colorectal tissue samples versus normal tissue. The microarray experiment is based on 16 control samples, 38 CRC plasma samples from colorectal cancer patients who have not undergone chemotherapy, and 17 chemo-treated samples. In the case of the analysis of CRC cancer versus healthy control we identified 359 altered miRNAs (214 downregulated and 60 upregulated), considering as the cutoff value a fold-change of ±1.5 and p < 0.01. An additional microarray analysis was performed on plasma from untreated colorectal cancer (n = 38) and chemotherapy-treated colorectal cancer patients (n = 17), which revealed 15 downregulated miRNAs and 53 upregulated miRNAs, demonstrating that the plasma miRNA pattern is affected by chemotherapy and emphasizing important regulators of drug resistance mechanisms. For the validation of the microarray data, we selected a panel of 4 miRNAs from the common miRNA signatures for colon and rectal cancer (miR-642b-3p, miR-195-5p and miR-4741). At the tissue level, the expression levels were in agreement with those observed in colorectal plasma. miR-1228-3p, the top upregulated miRNA in CRC, was chosen to be validated on tissue and plasma samples, as it was demonstrated to be downregulated at tissue level in our patient cohort. This was confirmed by TCGA data and was one example of ta ranscript that has a different expression level between tumor tissue and plasma. Developing more efficient investigation methods will help explain the mechanisms responsible for miRNAs released in biofluids, which is the most upregulated transcript in colorectal plasma samples and which can function as a prediction tool within the oncological field.

Nanoscale delivery systems for microRNAs in cancer therapy.

Concomitant with advances in research regarding the role of miRNAs in sustaining carcinogenesis, major concerns about their delivery options for anticancer therapies have been raised. The answer to this problem may come from the world of nanoparticles such as liposomes, exosomes, polymers, dendrimers, mesoporous silica nanoparticles, quantum dots and metal-based nanoparticles which have been proved as versatile and valuable vehicles for many biomolecules including miRNAs. In another train of thoughts, the general scheme of miRNA modulation consists in inhibition of oncomiRNA expression and restoration of tumor suppressor ones. The codelivery of two miRNAs or miRNAs in combination with chemotherapeutics or small molecules was also proposed. The present review presents the latest advancements in miRNA delivery based on nanoparticle-related strategies.

The Role of Nrf2 Activity in Cancer Development and Progression.

Nrf2 is a transcription factor that stimulates the expression of genes which have antioxidant response element-like sequences in their promoter. Nrf2 is a cellular protector, and this principle applies to both normal cells and malignant cells. While healthy cells are protected from DNA damage induced by reactive oxygen species, malignant cells are defended against chemo- or radiotherapy. Through our literature search, we found that Nrf2 activates several oncogenes unrelated to the antioxidant activity, such as Matrix metallopeptidase 9 (MMP-9), B-cell lymphoma 2 (BCL-2), B-cell lymphoma-extra large (BCL-xL), Tumour Necrosis Factor α (TNF-α), and Vascular endothelial growth factor A (VEGF-A). We also did a brief analysis of The Cancer Genome Atlas (TCGA) data of lung adenocarcinoma concerning the effects of radiation therapy and found that the therapy-induced Nrf2 activation is not universal. For instance, in the case of recurrent disease and radiotherapy, we observed that, for the majority of Nrf2-targeted genes, there is no change in expression level. This proves that the universal, axiomatic rationale that Nrf2 is activated as a response to chemo- and radiation therapy is wrong, and that each scenario should be carefully evaluated with the help of Nrf2-targeted genes. Moreover, there were nine genes involved in lipid peroxidation, which showed underexpression in the case of new radiation therapy: ADH1A, ALDH3A1, ALDH3A2, ADH1B, GPX2, ADH1C, ALDH6A1, AKR1C3, and NQO1. This may relate to the fact that, while some studies reported the co-activation of Nrf2 and other oncogenic signaling pathways such as Phosphoinositide 3-kinases (PI3K), mitogen-activated protein kinase (MAPK), and Notch1, other reported the inverse correlation between Nrf2 and the tumor-promoter Transcription Factor (TF), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Lastly, Nrf2 establishes its activity through interactions at multiple levels with various microRNAs. MiR-155, miR-144, miR-28, miR-365-1, miR-93, miR-153, miR-27a, miR-142, miR-29-b1, miR-340, and miR-34a, either through direct repression of Nrf2 messenger RNA (mRNA) in a Kelch-like ECH-associated protein 1 (Keap1)-independent manner or by enhancing the Keap1 cellular level, inhibit the Nrf2 activity. Keap1-Nrf2 interaction leads to the repression of miR-181c, which is involved in the Nuclear factor kappa light chain enhancer of activated B cells (NF-κB) signaling pathway. Nrf2's role in cancer prevention, diagnosis, prognosis, and therapy is still in its infancy, and the future strategic planning of Nrf2-based oncological approaches should also consider the complex interaction between Nrf2 and its various activators and inhibitors.

Connecting the dots between different networks: miRNAs associated with bladder cancer risk and progression.

BACKGROUND: Bladder cancer (BC) is a common urothelial malignancy, characterized by a high recurrence rate. The biology of bladder cancer is complex and needs to be deciphered. The latest evidence reveals the critical role of the non-coding RNAs, particularly microRNAs (miRNAs), as vital regulatory elements in cancer. METHOD: We performed a miRNAs microarray using paired tissues (tumor and adjacent normal bladder tissue), followed by the validation with qRT-PCR of five selected transcripts. Additional next-generation sequencing investigation established the interconnection among the altered miRNAs and mutated genes. Based on the overlapping between TCGA data and data obtained in the study, we focused on the systematic identification of altered miRNAs and genes mutated involved in bladder cancer tumorigenesis and progression. RESULTS: By overlapping the miRNAs expression data, the two patient cohorts, we identified 18 miRNAs downregulated and, 187 miRNAs upregulated. qRT-PCR validation was completed using a selected panel of two downregulated (miR-139-5p and miR-143-5p) and three up-regulated miRNAs (miR-141b, miR-200 s or miR-205). Altered miRNAs patterns are interrelated to bladder tumorigenesis, allowing them to be used for the development of novel diagnostic and prognostic biomarkers. Three EMT-related upregulated miRNAs have an essential role in the molecular mechanisms, specifically key processes underlying tumorigenesis, invasion and metastasis. Using the Ampliseq Cancer Panel kit and Ion Torrent PGM Next-Generation Sequencing an increased mutation rate for TP53, FGFR3, KDR, PIK3CA and ATM were observed, but the mutational status for only TP53 was correlated to the survival rate. The miRNAs pattern, along with the gene mutation pattern attained, can assist for better patient diagnosis. CONCLUSION: This study thereby incorporates miRNAs as critical players in bladder cancer prognosis, where their altered gene expression profiles have a critical biological function in relationship with tumor molecular phenotype. The miRNA-mRNA regulatory networks identified in BC are ripe for exploitation as biomarkers or targeted therapeutic strategies.

SERS-based differential diagnosis between multiple solid malignancies: breast, colorectal, lung, ovarian and oral cancer.

PURPOSE: Surface-enhanced Raman scattering (SERS) spectroscopy on serum and other biofluids for cancer diagnosis represents an emerging field, which has shown promising preliminary results in several types of malignancies. The purpose of this study was to demonstrate that SERS spectroscopy on serum can be employed for the differential diagnosis between five of the leading malignancies, ie, breast, colorectal, lung, ovarian and oral cancer. PATIENTS AND METHODS: Serum samples were acquired from healthy volunteers (n=39) and from patients diagnosed with breast (n=42), colorectal (n=109), lung (n=33), oral (n=17), and ovarian cancer (n=13), comprising n=253 samples in total. SERS spectra were acquired using a 532 nm laser line as excitation source, while the SERS substrates were represented by Ag nanoparticles synthesized by reduction with hydroxylamine. The classification accuracy yielded by SERS was assessed by principal component analysis-linear discriminant analysis (PCA-LDA). RESULTS: The sensitivity and specificity in discriminating between cancer patients and controls was 98% and 91%, respectively. Cancer samples were correctly assigned to their corresponding cancer types with an accuracy of 88% for oral cancer, 86% for colorectal cancer, 80% for ovarian cancer, 76% for breast cancer and 59% for lung cancer. CONCLUSION: SERS on serum represents a promising strategy of diagnosing cancer which can discriminate between cancer patients and controls, as well as between cancer types such as breast, colorectal, lung ovarian and oral cancer.

Role of Key Micronutrients from Nutrigenetic and Nutrigenomic Perspectives in Cancer Prevention.

Regarding cancer as a genetic multi-factorial disease, a number of aspects need to be investigated and analyzed in terms of cancer's predisposition, development and prognosis. One of these multi-dimensional factors, which has gained increased attention in the oncological field due to its unelucidated role in risk assessment for cancer, is diet. Moreover, as studies advance, a clearer connection between diet and the molecular alteration of patients is becoming identifiable and quantifiable, thereby replacing the old general view associating specific phenotypical changes with the differential intake of nutrients. Respectively, there are two major fields concentrated on the interrelation between genome and diet: nutrigenetics and nutrigenomics. Nutrigenetics studies the effects of nutrition at the gene level, whereas nutrigenomics studies the effect of nutrients on genome and transcriptome patterns. By precisely evaluating the interaction between the genomic profile of patients and their nutrient intake, it is possible to envision a concept of personalized medicine encompassing nutrition and health care. The list of nutrients that could have an inhibitory effect on cancer development is quite extensive, with evidence in the scientific literature. The administration of these nutrients showed significant results in vitro and in vivo regarding cancer inhibition, although more studies regarding administration in effective doses in actual patients need to be done.

Overview upon miR-21 in lung cancer: focus on NSCLC.

Considering the high mortality rate encountered in lung cancer, there is a strong need to explore new biomarkers for early diagnosis and also improved therapeutic targets to overcome this issue. The implementation of microRNAs as important regulators in cancer and other pathologies expanded the possibilities of lung cancer management and not only. MiR-21 represents an intensively studied microRNA in many types of cancer, including non-small cell lung cancer (NSCLC). Its role as an oncogene is underlined in multiple studies reporting the upregulated expression of this sequence in patients diagnosed with this malignancy; moreover, several studies associated this increased expression of miR-21 with a worse outcome within NSCLC patients. The same pattern is supported by the data existent in the Cancer Genome Atlas (TCGA). The carcinogenic advantage generated by miR-21 in NSCLC resides in the target genes involved in multiple pathways such as cell growth and proliferation, angiogenesis, invasion and metastasis, but also chemo- and radioresistance. Therapeutic modulation of miR-21 by use of antisense sequences entrapped in different delivery systems has shown promising results in impairment of NSCLC. Hereby, we review the mechanisms of action of miR-21 in cancer and the associated changes upon tumor cells together a focused perspective on NSCLC signaling, prognosis and therapy.

Differential Effect of Smoking on Gene Expression in Head and Neck Cancer Patients.

Smoking is a well-known behavior that has an important negative impact on human health, and is considered to be a significant factor related to the development and progression of head and neck squamous cell carcinomas (HNSCCs). Use of high-dimensional datasets to discern novel HNSCC driver genes related to smoking represents an important challenge. The Cancer Genome Atlas (TCGA) analysis was performed in three co-existing groups of HNSCC in order to assess whether gene expression landscape is affected by tobacco smoking, having quit, or non-smoking status. We identified a set of differentially expressed genes that discriminate between smokers and non-smokers or based on human papilloma virus (HPV)16 status, or the co-occurrence of these two exposome components in HNSCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways classification shows that most of the genes are specific to cellular metabolism, emphasizing metabolic detoxification pathways, metabolism of chemical carcinogenesis, or drug metabolism. In the case of HPV16-positive patients it has been demonstrated that the altered genes are related to cellular adhesion and inflammation. The correlation between smoking and the survival rate was not statistically significant. This emphasizes the importance of the complex environmental exposure and genetic factors in order to establish prevention assays and personalized care system for HNSCC, with the potential for being extended to other cancer types.

The silent healer: miR-205-5p up-regulation inhibits epithelial to mesenchymal transition in colon cancer cells by indirectly up-regulating E-cadherin expression.

EMT represents the dominant program within advanced stages of colon cancer, where cells acquire migratory characteristics in order to invade secondary tissues and form metastasis. Where the majority of the therapeutic strategies are concentrated on the reduction of the tumor mass through different apoptotic mechanisms, the present study advocates an important role for miR-205-5p in impairment of colon cancer cells migration and restoration of the epithelial phenotype. Upon identification of a homogenous downregulated profile for miR-205-5p in colon adenocarcinoma patients, functional studies demonstrated that experimental upregulation of this sequence is able to significantly raise the levels of E-cadherin through direct inhibition of ZEB1. Moreover, the elevation in CDH1 expression was translated into functional parameters where cells lost their invasion and migratory characteristics and formed homogenous clusters through adhesion interactions. Survival analysis of colon adenocarcinoma patients revealed that low levels of miR-205-5p are associated with an unfavorable prognostic compared to those with increased expression, demonstrating the possible clinical utility of miR-205-5p replacement. Exogenous administration of miRNA mimics was not associated with significant changes in cell viability or inflammatory pathways. Therefore, the proposed strategy is aiming towards inhibition of metastasis and limitation of the tumor borders in advanced stages patients in order to prolong the survival time and to increase the efficiency of the current therapeutic strategies.

Fibroblast dynamics as an in vitro screening platform for anti-fibrotic drugs in primary myelofibrosis.

Although the cause for bone marrow fibrosis in patients with myelofibrosis remains controversial, it has been hypothesized that it is caused by extensive fibroblast proliferation under the influence of cytokines generated by the malignant megakaryocytes. Moreover, there is no known drug therapy which could reverse the process. We studied the fibroblasts in a novel system using the hanging drop method, evaluated whether the fibroblasts obtain from patients are part of the malignant clone of not and, using this system, we screen a large library of FDA-approved drugs to identify potential drugs candidates that might be useful in the treatment of this disease, specifically which would inhibit fibroblast proliferation and the development of bone marrow fibrosis. We have found that the BM fibroblasts are not part of the malignant clone, as previously suspected and two immunosuppressive medications-cyclosporine and mycophenolate mophetil, as most potent suppressors of the fibroblast collagen production thus potentially inhibitors of bone marrow fibrosis production in myelofibrosis.

The use of rotation to fentanyl in cancer-related pain.

Pain is commonly diagnosed with respect to cancer and heart diseases, being a major symptom in most neoplastic diseases. Uncontrolled pain leads to a decrease in the quality of life and an increase in the morbidity of the patient. Opioids represent the best analgetic supportive therapy and are frequently used in patients suffering from cancer and experiencing a high level of pain. Opioid treatment starts with a gradual titration of the dose until the minimum effective dose and the maximum tolerated dose are determined. Opioid rotation refers to the switch from one opioid to another in order to get a better response to analgetic therapy and reduce side effects. Fentanyl therapy is recommended to be continued during chemotherapy, radiotherapy, or in the case of surgical intervention. Rotation to fentanyl patches is an efficient and elegant solution for cancer patients, with reduced side effects. Opioid rotation, especially to fentanyl, was shown to increase the quality of life in patients with malignant disease. Finally, rotation to fentanyl is also advantageous from an economic point of view.