Helena Cirenajwis

The Antiproliferative Effect of Dietary Fiber Phenolic Compounds Ferulic Acid and p-Coumaric Acid on the Cell Cycle of Caco-2 Cells.

Epidemiological and animal studies have shown that dietary fiber is protective against the development of colon cancer. Dietary fiber is a rich source of the hydroxycinnamic acids ferulic acid (FA) and p-coumaric acid (p-CA), which both may contribute to the protective effect. We have investigated the effects of FA and p-CA treatment on global gene expression in Caco-2 colon cancer cells. The Caco-2 cells were treated with 150 μM FA or p-CA for 24 h, and gene expression was analyzed with cDNA microarray technique. A total of 517 genes were significantly affected by FA and 901 by p-CA. As we previously have found that FA or p-CA treatment delayed cell cycle progression, we focused on genes involved in proliferation and cell cycle regulation. The expressions of a number of genes involved in centrosome assembly, such as RABGAP1 and CEP2, were upregulated by FA treatment as well as the gene for the S phase checkpoint protein SMC1L1. p-CA treatment upregulated CDKN1A expression and downregulated CCNA2, CCNB1, MYC, and ODC1. Some proteins corresponding to the affected genes were also studied. Taken together, the changes found can partly explain the effects of FA or p-CA treatment on cell cycle progression, specifically in the S phase by FA and G2/M phase by p-CA treatment.

The clinical significance of BRAF and NRAS mutations in a clinic‐based metastatic melanoma cohort

BRAF and NRAS mutations are frequently found in melanoma tumours, and recently developed BRAF‐targeted therapies demonstrate significant clinical benefit.

Multiregion whole-exome sequencing uncovers the genetic evolution and mutational heterogeneity of early-stage metastatic melanoma

Cancer genome sequencing has shed light on the underlying genetic aberrations that drive tumorigenesis. However, current sequencing-based strategies, which focus on a single tumor biopsy, fail to take into account intratumoral heterogeneity. To address this challenge and elucidate the evolutionary history of melanoma, we performed whole-exome and transcriptome sequencing of 41 multiple melanoma biopsies from eight individual tumors. This approach revealed heterogeneous somatic mutations in the range of 3%-38% in individual tumors. Known mutations in melanoma drivers BRAF and NRAS were always ubiquitous events. Using RNA sequencing, we found that the majority of mutations were not expressed or were expressed at very low levels, and preferential expression of a particular mutated allele did not occur frequently. In addition, we found that the proportion of ultraviolet B (UVB) radiation-induced C>T transitions differed significantly (P < 0.001) between early and late mutation acquisition, suggesting that different mutational processes operate during the evolution of metastatic melanoma. Finally, clinical history reports revealed that patients harboring a high degree of mutational heterogeneity were associated with more aggressive disease progression. In conclusion, our multiregion tumor-sequencing approach highlights the genetic evolution and non-UVB mutational signatures associated with melanoma development and progression, and may provide a more comprehensive perspective of patient outcome. Cancer Res; 76(16); 4765-74. ©2016 AACR.

Genome-wide DNA methylation analysis in melanoma reveals the importance of CpG methylation in MITF regulation

The microphthalmia-associated transcription factor (MITF) is a key regulator of melanocyte development and a lineage-specific oncogene in melanoma; a highly lethal cancer known for its unpredictable clinical course. MITF is regulated by multiple intracellular signaling pathways, although the exact mechanisms that determine MITF expression and activity remain incompletely understood. In this study, we obtained genome-wide DNA methylation profiles from 50 stage IV melanomas, normal melanocytes, keratinocytes, and dermal fibroblasts and utilized The Cancer Genome Atlas data for experimental validation. By integrating DNA methylation and gene expression data, we found that hypermethylation of MITF and its co-regulated differentiation pathway genes corresponded to decreased gene expression levels. In cell lines with a hypermethylated MITF-pathway, overexpression of MITF did not alter the expression level or methylation status of the MITF pathway genes. In contrast, however, demethylation treatment of these cell lines induced MITF-pathway activity, confirming that gene regulation was controlled via methylation. The discovery that the activity of the master regulator of pigmentation, MITF, and its downstream targets may be regulated by hypermethylation has significant implications for understanding the development and evolvement of melanoma.

Reduction of the putative CD44+CD24- breast cancer stem cell population by targeting the polyamine metabolic pathway with PG11047.

Cancer stem cells (CSCs) are considered to be of particular concern in cancer as they possess inherent properties of self-renewal and differentiation, along with expressing certain genes related to a mesenchymal phenotype. These features favour the promotion of tumour recurrence and metastasis in cancer patients. Thus, the optimal chemotherapeutic treatment should target the CSC population, either by killing these cells and/or by inducing their transition to a more differentiated epithelial-like phenotype. Experiments were carried out on the trastuzumab-resistant human epidermal growth factor receptor 2-overexpressing breast cancer cell line JIMT-1 to unravel the chemotherapeutic effects of the polyamine analogue [1N,12N]bis(ethyl)-cis-6,7-dehydrospermine (PG11047) and of the polyamine biosynthetic inhibitor 2-difluoromethylornithine (DFMO) on the CD44+CD24− CSC population. Furthermore, effects on the properties of self-renewal and epithelial/mesenchymal markers were also investigated. Treatment with PG11047 reduced the CD44+CD24− subpopulation of JIMT-1 cells by approximately 50%, inhibited and/or reduced self-renewal capability of the CSC population, decreased cell motility and induced expression of mesenchymal to epithelial transition-associated proteins that are involved in promoting an epithelial phenotype. By contrast, DFMO slightly increased the CD44+CD24− subpopulation, increased cell motility and the level of mesenchymal-related proteins. DFMO treatment reduced the self-renewal capability of the CSC population. Both PG11047 and DFMO reduced the expression of the human epidermal growth factor receptor 2 protein, which is correlated to malignancy and resistance to trastuzumab in JIMT-1 cells. Our findings indicate that treatment with PG11047 targeted the CSC population by interfering with several stem cell-related properties, such as self-renewal, differentiation, motility and the mesenchymal phenotype.

Digital Holography and Cell Studies

Digital holographic microscopy (DHM) is a novel high-resolution imaging technique that offers real-time imaging and quantitative measurements of physiological parameters. It has developed into a broad field, and one of many interesting applications is to study cells without staining or labeling them and without affecting them in any way. Digital holography makes it possible to easily measure cell properties that previously have been very difficult to study in living cells, such as cell thickness, volume, and cell refractive index (Marquet et al., 2005; Rappaz et al. 2005; Molder et al., 2008; El-Schish et al., in press; Persson et al., in press). Living, dying or dead cells as well as fixed cells can be studied. The first DHM images showing living cells were published in 2003 and 2004 (You et al., 2003; Carl et al., 2004), making this field of research rather new. Two of the most interesting functions of DHM is 3-D imaging of objects and to make in-focus measurements over time. Digital holography has been used to study a wide range of cells, e.g. protozoa, bacteria and plant cells as well as several types of mammalian cells such as nerve cells and tumor cells (Emery et al., 2007; Kemper et al., 2006; Moon and Javidi 2007). It has also been applied for studies of cell proliferation, cell movement and cell morphology (Kemper et al., 2009; Yu et al., 2009). Movement in both 2-D and 3-D has been studied (Langehanenberg et al., 2009; Persson et al., in press). In addition, cell viability status can be determined using DHM (Kemper et al., 2006; Kemmler et al., 2007). Interestingly, it is possible to study both single cells and entire populations simultaneously, allowing for very detailed studies. In this chapter we will compare DHM with previously used techniques and discuss the benefits and drawbacks of digital holography cell measurements. We will also present cell studies made possible by DHM.

NF1-mutated melanoma tumors harbor distinct clinical and biological characteristics

In general, melanoma can be considered as a UV‐driven disease with an aggressive metastatic course and high mutational load, with only few tumors (acral, mucosal, and uveal melanomas) not induced by sunlight and possessing a lower mutational load. The most commonly activated pathway in melanoma is the mitogen‐activated protein kinase (MAPK) pathway. However, the prognostic significance of mutational stratification is unclear and needs further investigation. Here, in silico we combined mutation data from 162 melanomas subjected to targeted deep sequencing with mutation data from three published studies. Tumors from 870 patients were grouped according to BRAF, RAS, NF1 mutation or triple‐wild‐type status and correlated with tumor and patient characteristics. We found that the NF1‐mutated subtype had a higher mutational burden and strongest UV mutation signature. Searching for co‐occurring mutated genes revealed the RASopathy genes PTPN11 and RASA2, as well as another RAS domain‐containing gene RASSF2 enriched in the NF1 subtype after adjustment for mutational burden. We found that a larger proportion of the NF1‐mutant tumors were from males and with older age at diagnosis. Importantly, we found an increased risk of death from melanoma (disease‐specific survival, DSS; HR, 1.9; 95% CI, 1.21–3.10; P = 0.046) and poor overall survival (OS; HR, 2.0; 95% CI, 1.28–2.98; P = 0.01) in the NF1 subtype, which remained significant after adjustment for age, gender, and lesion type (DSS P = 0.03, OS P = 0.06, respectively). Melanoma genomic subtypes display different biological and clinical characteristics. The poor outcome observed in the NF1 subtype highlights the need for improved characterization of this group.

A role for antizyme inhibitor in cell proliferation

The polyamines are important for a variety of cellular functions, including cell growth. Their intracellular concentrations are controlled by a complex network of regulatory mechanisms, in which antizyme (Az) has a key role. Az reduces the cellular polyamine content by down-regulating both the enzyme catalysing polyamine biosynthesis, ornithine decarboxylase (ODC), and the uptake of polyamines. The activity of Az is repressed by the binding of a protein, named Az inhibitor (AzI), which is an enzymatically inactive homologue of ODC. Two forms of AzI have been described: AzI1, which is ubiquitous, and AzI2 which is expressed in brain and testis. In the present study, we have investigated the role of AzI1 in polyamine homeostasis and cell proliferation in breast cancer cells. The results obtained showed that the cellular content of AzI increased transiently after induction of cell proliferation by diluting cells in fresh medium. Inhibition of polyamine biosynthesis induced an even larger increase in the cellular AzI content, which remained significantly elevated during the 7-day experimental period. However, this increase was not a consequence of changes in cell cycle progression, as demonstrated by flow cytometry. Instead, the increase appeared to correlate with the cellular depletion of polyamines. Moreover, induced overexpression of AzI resulted in an increased cell proliferation with a concomitant increase in ODC activity and putrescine content. During mitosis, AzI1 was localised in a pattern that resembled that of the two centrosomes, confirming earlier observations. Taken together, the results indicate that AzI fulfils an essential regulatory function in polyamine homeostasis and cell proliferation.

Loss of CITED1, an MITF regulator, drives a phenotype switch in vitro and can predict clinical outcome in primary melanoma tumours

CITED1 is a non-DNA binding transcriptional co-regulator whose expression can distinguish the ‘proliferative’ from ‘invasive’ signature in the phenotype-switching model of melanoma. We have found that, in addition to other ‘proliferative’ signature genes, CITED1 expression is repressed by TGFβ while the ‘invasive’ signature genes are upregulated. In agreement, CITED1 positively correlates with MITF expression and can discriminate the MITF-high/pigmentation tumour molecular subtype in a large cohort (120) of melanoma cell lines. Interestingly, CITED1 overexpression significantly suppressed MITF promoter activation, mRNA and protein expression levels while MITF was transiently upregulated following siRNA mediated CITED1 silencing. Conversely, MITF siRNA silencing resulted in CITED1 downregulation indicating a reciprocal relationship. Whole genome expression analysis identified a phenotype shift induced by CITED1 silencing and driven mainly by expression of MITF and a cohort of MITF target genes that were significantly altered. Concomitantly, we found changes in the cell-cycle profile that manifest as transient G1 accumulation, increased expression of CDKN1A and a reduction in cell viability. Additionally, we could predict survival outcome by classifying primary melanoma tumours using our in vitro derived ‘CITED1-silenced’ gene expression signature. We hypothesize that CITED1 acts a regulator of MITF, functioning to maintain MITF levels in a range compatible with tumourigenesis.