Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules

Genetic and Genomic Pathways of Melanoma Development, Invasion and Metastasis

Melanoma is a serious form of skin cancer that accounts for 80% of skin cancer deaths. Recent studies have suggested that melanoma invasiveness is attributed to phenotype switching, which is a reversible type of cell behaviour with similarities to epithelial to mesenchymal transition. Phenotype switching in melanoma is reported to be independent of genetic alterations, whereas changes in gene transcription, and epigenetic alterations have been associated with invasiveness in melanoma cell lines. Here, we review mutational, transcriptional, and epigenomic alterations that contribute to tumour heterogeneity in melanoma, and their potential to drive melanoma invasion and metastasis. We also discuss three models that are hypothesized to contribute towards aspects of tumour heterogeneity and tumour progression in melanoma, namely the clonal evolution model, the cancer stem cell model, and the phenotype switching model. We discuss the merits and disadvantages of each model in explaining tumour heterogeneity in melanoma, as a precursor to invasion and metastasis

Genome-wide DNA Methylation and Gene Expression Analysis of Non-invasive and Invasive Melanoma Cell Lines

Melanoma is a serious form of skin cancer derived from the pigment producing cells called melanocytes. New Zealand has the highest rate of melanoma in the world with as high as 50 cases of melanoma per 100,000 people, rendering melanoma as the fourth most common cancer. Melanoma can be cured if detected in the early stages, but the metastatic form of melanomas has a poor survival rate. Although advanced therapies have improved the patient survival rate, drug resistance is still a major problem in metastatic melanoma. It has become increasingly evident that the major obstacle in melanoma therapy is its heterogeneous nature. Metastatic tumour cells in melanoma are known to switch back and forth from a non-invasive to an invasive state, due to tumour microenvironmental cues, such as hypoxia. Consequently, it is difficult to target the subpopulations of invasive cells that escape therapies by switching back to a non-invasive state, and then later initiate the invasive properties by switching back to the invasive state. Therefore, a better understanding of the underlying mechanisms that initiate this phenotype switch is required. Previous studies carried out by our group and other investigators have demonstrated two main phenotypes in melanoma that were distinct in their gene expression profiles, and which were independent of the tumour-associated genetic mutations from which these cell lines were derived. The melanoma cells can switch back and forth from one phenotype to the other, with the non-invasive phenotype demonstrating a low invasive capacity, and invasive phenotype demonstrating a high invasive capacity. Given that these phenotypes are reversible and independent of the mutational status, it is plausible that an epigenetic mechanism is involved in the regulation of these phenotypes. Epigenetic regulation is defined as the mechanism that transduces the inheritance of gene expression without altering the underlying DNA sequence. DNA methylation is an epigenetic mechanism which involves addition of a methyl group to the cytosine base. In somatic tissues, the DNA methylation profile underlies cellular identity which is conserved over a lifetime. Changes in the environment are capable of altering the DNA methylation which consequently leads to changes in the phenotype. I hypothesized that DNA methylation, which is the most stable and reversible epigenetic mechanism, would underlie the mechanism that distinguishes the invasive from non-invasive melanomas. To address this hypothesis, a genome-wide DNA methylation analysis was carried out using reduced representation bisulphite sequencing (RRBS). Differential methylation analysis revealed 39 differentially methylated fragments (DMFs) in the invasive cell lines as compared to the non-invasive cell lines, with 20 DMFs hypermethylated and 19 DMFs hypomethylated in the invasive melanomas. This observation confirmed that DNA methylation profiles can distinguish the invasive group from the non-invasive group of melanoma cell lines. To identify the relationship between DNA methylation and gene expression, genome-wide RNA sequencing (RNA-Seq) was carried out on the same cell lines. The RNA-Seq results identified interesting genes which were associated with growth factor binding, transmembrane receptor kinase activity, cell adhesion and extra cellular matrix organization. Comparison of the DMFs identified from multiple RRBS analyses with differentially expressed genes identified by multiple RNA-Seq analyses resulted in a total of 17 common differentially expressed genes associated with DNA methylation. Of these 17 genes, AVPI1 was selected for further validation because it had significant promoter hypermethylation in the invasive cell lines compared to non-invasive cell lines. In addition, low expression of this gene was identified in the invasive cell lines, and this was associated with MAPK activity and epithelial sodium channel (ENaC) downregulation. Functional analysis suggested that AVPI1 promoter methylation may be associated indirectly with melanoma invasion through an intermediate mechanism of ENaC regulation and epithelial mesenchymal transition (EMT). Overall, this study has provided novel insights into distinct DNA methylation profiles between invasive and non-invasive melanoma cell lines, which predominantly included cell lines derived from New Zealand metastatic melanoma patients. Integrative analysis of DNA methylation and gene expression revealed potential genes involved in the invasive phenotype of melanoma, which was followed by functional analysis of AVPI1 to investigate its role in the invasive phenotype. These investigations of AVPI1 and other genes identified from the integrative analysis of DNA methylation and gene expression have provided a platform, which can further be used to establish their role in phenotype switching

Genome-wide DNA Methylation and Gene Expression Analysis of Non-invasive and Invasive Melanoma Cell Lines

Melanoma is a serious form of skin cancer derived from the pigment producing cells called melanocytes. New Zealand has the highest rate of melanoma in the world with as high as 50 cases of melanoma per 100,000 people, rendering melanoma as the fourth most common cancer. Melanoma can be cured if detected in the early stages, but the metastatic form of melanomas has a poor survival rate. Although advanced therapies have improved the patient survival rate, drug resistance is still a major problem in metastatic melanoma. It has become increasingly evident that the major obstacle in melanoma therapy is its heterogeneous nature. Metastatic tumour cells in melanoma are known to switch back and forth from a non-invasive to an invasive state, due to tumour microenvironmental cues, such as hypoxia. Consequently, it is difficult to target the subpopulations of invasive cells that escape therapies by switching back to a non-invasive state, and then later initiate the invasive properties by switching back to the invasive state. Therefore, a better understanding of the underlying mechanisms that initiate this phenotype switch is required. Previous studies carried out by our group and other investigators have demonstrated two main phenotypes in melanoma that were distinct in their gene expression profiles, and which were independent of the tumour-associated genetic mutations from which these cell lines were derived. The melanoma cells can switch back and forth from one phenotype to the other, with the non-invasive phenotype demonstrating a low invasive capacity, and invasive phenotype demonstrating a high invasive capacity. Given that these phenotypes are reversible and independent of the mutational status, it is plausible that an epigenetic mechanism is involved in the regulation of these phenotypes. Epigenetic regulation is defined as the mechanism that transduces the inheritance of gene expression without altering the underlying DNA sequence. DNA methylation is an epigenetic mechanism which involves addition of a methyl group to the cytosine base. In somatic tissues, the DNA methylation profile underlies cellular identity which is conserved over a lifetime. Changes in the environment are capable of altering the DNA methylation which consequently leads to changes in the phenotype. I hypothesized that DNA methylation, which is the most stable and reversible epigenetic mechanism, would underlie the mechanism that distinguishes the invasive from non-invasive melanomas. To address this hypothesis, a genome-wide DNA methylation analysis was carried out using reduced representation bisulphite sequencing (RRBS). Differential methylation analysis revealed 39 differentially methylated fragments (DMFs) in the invasive cell lines as compared to the non-invasive cell lines, with 20 DMFs hypermethylated and 19 DMFs hypomethylated in the invasive melanomas. This observation confirmed that DNA methylation profiles can distinguish the invasive group from the non-invasive group of melanoma cell lines. To identify the relationship between DNA methylation and gene expression, genome-wide RNA sequencing (RNA-Seq) was carried out on the same cell lines. The RNA-Seq results identified interesting genes which were associated with growth factor binding, transmembrane receptor kinase activity, cell adhesion and extra cellular matrix organization. Comparison of the DMFs identified from multiple RRBS analyses with differentially expressed genes identified by multiple RNA-Seq analyses resulted in a total of 17 common differentially expressed genes associated with DNA methylation. Of these 17 genes, AVPI1 was selected for further validation because it had significant promoter hypermethylation in the invasive cell lines compared to non-invasive cell lines. In addition, low expression of this gene was identified in the invasive cell lines, and this was associated with MAPK activity and epithelial sodium channel (ENaC) downregulation. Functional analysis suggested that AVPI1 promoter methylation may be associated indirectly with melanoma invasion through an intermediate mechanism of ENaC regulation and epithelial mesenchymal transition (EMT). Overall, this study has provided novel insights into distinct DNA methylation profiles between invasive and non-invasive melanoma cell lines, which predominantly included cell lines derived from New Zealand metastatic melanoma patients. Integrative analysis of DNA methylation and gene expression revealed potential genes involved in the invasive phenotype of melanoma, which was followed by functional analysis of AVPI1 to investigate its role in the invasive phenotype. These investigations of AVPI1 and other genes identified from the integrative analysis of DNA methylation and gene expression have provided a platform, which can further be used to establish their role in phenotype switching

ECHO…for a change!!

The childhood obesity is increased more than three folds in last two decades in developed world. There is nutritional transition seen in the developing world including India. The westernization in diet of the Indian population along with prosperity brings the brunt of overweight and obesity. This has future implications of liver diseases, heart diseases, hypertension, hyperlipidaemia, insulin resistance; malignancies. Mumbai is the prosperous city and an economical capital of India. Also, the rampant use junk food, common outdoor eating’s, no grounds to play for children make the high likelihood that the prevalence of obesity to be higher than rest of the country. It can profoundly affect children’s physical health, social, and emotional well-being and self-esteem. It is also associated with poor academic performance and a lower quality of life experienced by the child. One of the best strategies to reduce childhood obesity is to improve the eating and exercise habits of the entire family. Treating and preventing childhood obesity helps protect the child’s health and has tremendous impact on child’s Physical and academic performance. And hence we at Aastha Bariatrics took initiative and launched ECHO... for a change (‘E’radicating ‘C’Hild ‘H’ood ‘O’besity), a pan Mumbai campaign against childhood obesity. This campaign was done in 15 high schools across Mumbai, which covered in total of 9000 students

The role of renal denervation in cardiology and beyond: An updated comprehensive review and future directives

Renal denervation (RDN) is a minimally invasive intervention performed by denervation of the nervous fibers in the renal plexus, which decreases sympathetic activity. These sympathetic nerves influence various physiological functions that regulate blood pressure (BP), including intravascular volume, electrolyte composition, and vascular tone. Although proven effective in some trials, controversial trials, such as the Controlled Trial of Renal Denervation for Resistant Hypertension (SYMPLICITY-HTN3), have demonstrated contradictory results for the effectiveness of RDN in resistant hypertension (HTN). In the treatment of HTN, individuals with primary HTN are expected to experience greater benefits compared to those with secondary HTN due to the diverse underlying causes of secondary HTN. Beyond its application for HTN, RDN has also found utility in addressing cardiac arrhythmias, such as atrial fibrillation, and managing cases of heart failure. Non-cardiogenic applications of RDN include reducing the intensity of obstructive sleep apnea (OSA), overcoming insulin resistance, and in chronic kidney disease (CKD) patients. This article aims to provide a comprehensive review of RDN and its uses in cardiology and beyond, along with providing future directions and perspectives

Marked Global DNA Hypomethylation Is Associated with Constitutive PD-L1 Expression in Melanoma

Summary: Constitutive expression of the immune checkpoint, PD-L1, inhibits anti-tumor immune responses in cancer, although the factors involved in PD-L1 regulation are poorly understood. Here we show that loss of global DNA methylation, particularly in intergenic regions and repeat elements, is associated with constitutive (PD-L1CON), versus inducible (PD-L1IND), PD-L1 expression in melanoma cell lines. We further show this is accompanied by transcriptomic up-regulation. De novo epigenetic regulators (e.g., DNMT3A) are strongly correlated with PD-L1 expression and methylome status. Accordingly, decitabine-mediated inhibition of global methylation in melanoma cells leads to increased PD-L1 expression. Moreover, viral mimicry and immune response genes are highly expressed in lymphocyte-negative plus PD-L1-positive melanomas, versus PD-L1-negative melanomas in The Cancer Genome Atlas (TCGA). In summary, using integrated genomic analysis we identified that global DNA methylation influences PD-L1 expression in melanoma, and hence melanoma's ability to evade anti-tumor immune responses. These results have implications for combining epigenetic therapy with immunotherapy. : Genetics; Genomics; Cancer; Transcriptomics Subject Areas: Genetics, Genomics, Cancer, Transcriptomic

Risk of leptomeningeal carcinomatosis in patients with brain metastases treated with stereotactic radiosurgery

© 2017, Springer Science+Business Media, LLC, part of Springer Nature. There is limited available literature examining factors that predispose patients to the development of LMC after stereotactic radiosurgery (SRS) for brain metastases. We sought to evaluate risk factors that may predispose patients to LMC after SRS treatment in this case–control study of patients with brain metastases who underwent single-fraction SRS between 2011 and 2016. Demographic and clinical information were collected retrospectively for 19 LMC cases and 30 controls out of 413 screened patients with brain metastases. Risk factors of interest were evaluated by univariate and multivariate logistic regression analyses and overall survival rates were evaluated by Kaplan–Meier survival analysis. About 5% of patients with brain metastases treated with SRS developed LMC. Patients with LMC (median 154 days, 95% CI 33–203 days) demonstrated a poorer overall survival than matched controls (median 417 days, 95% CI 121–512 days, p = 0.002). The most common primary tumor histologies that lead to the development of LMC were non-small cell lung cancer (36.8%), breast cancer (26.3%), and melanoma (21.1%). No association was found between the risk of LMC and the location of the brain lesion or total volume of brain metastases. Prior surgical resection of brain metastases before SRS was associated with a 6.5 times higher odds (95% CI 1.45–29.35, p = 0.01) of developing LMC post-radiosurgery compared to those with no prior resections of brain metastases. Additionally, adjuvant WBRT may help to reduce the risk of LMC and can be considered in decision-making for patients who have had brain metastasectomy

Differential Expression of BARD1 Isoforms in Melanoma

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted