Melanoma Transition Is Frequently Accompanied by a Loss of Cytoglobin Expression in Melanocytes: A Novel Expression Site of Cytoglobin

<div><p>The tissue distribution and function of hemoglobin or myoglobin are well known; however, a newly found cytoglobin (CYGB), which also belongs to the globin family, remains to be characterized. To assess its expression in human malignancies, we sought to screen a number of cell lines originated from many tissues using northern blotting and real time PCR techniques. Unexpectedly, we found that several, but not all, melanoma cell lines expressed CYGB mRNA and protein at much higher levels than cells of other origins. Melanocytes, the primary origin of melanoma, also expressed CYGB at a high level. To verify these observations, immunostaining and immunoblotting using anti-CYGB antibody were also performed. Bisulfite-modified genomic sequencing revealed that several melanoma cell lines that abrogated CYGB expression were found to be epigenetically regulated by hypermethylation in the promoter region of <i>CYGB</i> gene. The RNA interference-mediated knockdown of the CYGB transcript in CYGB expression-positive melanoma cell lines resulted in increased proliferation <i>in vitro</i> and <i>in vivo</i>. Flow cytometric analysis using 2′-, 7′-dichlorofluorescein diacetate (DCFH-DA), an indicator of reactive oxygen species (ROS), revealed that the cellular ROS level may be involved in the proliferative effect of CYGB. Thus, CYGB appears to play a tumor suppressive role as a ROS regulator, and its epigenetic silencing, as observed in CYGB expression-negative melanoma cell lines, might function as an alternative pathway in the melanocyte-to-melanoma transition.</p></div

CYGB protein is overexpressed in melanocytes and some of its malignant offspring.

<p><b>(A)</b> Immunoblot analysis of CYGB protein (indicated by an arrow) in NHDF, keratinocytes and melanocytes from skin and 8 melanoma cell lines (WM35 to HS294T). The image was obtained using ImageQuant LAS 3000 with an exposure time of 15 sec. The minor band, possibly a degradation product, is observed below the major band, which is prominent in melanocytes. The molecular mass marker (kDa) is given on the left side. β-actin was used as a loading control. <b>(B)</b> Immunohistochemical analysis of formalin-fixed, paraffin-embedded human normal skin using PNL2 (melanocyte marker) and CYGB antibodies. Two different regions (a) and (b) stained using each antibody are shown: (a) 4 × magnification, scale bar = 100 μm. (b) 20× magnification, scale bar  = 10 μm.</p

Sequencing histograms for the CpG island of the CYGB promoter region.

<p>Of the 24 CpG sites known to be methylated in the CYGB promoter region, 9 sites analyzed for methylation are shown. Cytosines methylated in A375 <b>(B)</b> are underlined. The corresponding cytosines are entirely unmethylated in melanocytes <b>(A)</b> and G361 <b>(C)</b>, resulting in the sequence “TpG” after bisulfite treatment.</p

<i>CYGB</i>-knocked down melanoma cells increase proliferation.

<p><b>(A)</b> Immunoblot data for C32TG and G361 cells transfected with CYGB siRNA or control siRNA. β-actin was used as a loading control. <b>(B)</b> Cellular proliferation pattern for G361 and C32TG cells transfected with CYGB siRNA (si_CYGB) and control siRNA (si_Control). The MTT analysis was performed daily (1d to 4d) post-transfection. The value represents the mean from three independent experiments; OD value, 570 nm. bars, SEM. * <i>P</i><0.05, ** <i>P</i><0.01. <b>(C)</b> Growth analysis of xenografted G361 tumors in nude mice. G361 cells expressing shRNA against CYGB or control shRNA were subcutaneously implanted into the interscapular region of five female mice. Tumor size was measured at the indicated time points. Bars, SEM. * <i>P</i><0.05.</p

CYGB protects G361 cells from H₂O₂-induced cell death.

<p>A flow cytometric analysis was performed to determine the ROS level <b>(A)</b> and apoptosis <b>(B)</b> in G361 cells transfected with a CYGB-siRNA or a control-siRNA. In <b>(A)</b>, 2′-, 7′-dichlorofluorescein diacetate (DCFH-DA) was used. CYGB-knocked down G361 cells treated with 3 mM N-acetyl-L-cystein (NAC) were also compared. <b>(B)</b> Annexin V and PI staining was done after exposure of the cells to 100 μM H₂O₂ for 0 h (−) or 24 h (+). At an early stage of apoptosis the cells bind to Annexin V while still excluding PI. At a late stage of apoptosis they bind to Annexin V and stain brightly with PI. (a) Control-siRNA-transfected, and (b) CYGB-siRNA-transfected G361 cells.</p

Concordance of plasma and tumor results for <i>RAS</i> and <i>BRAF</i> mutations.

<p>Concordance of plasma and tumor results for <i>RAS</i> and <i>BRAF</i> mutations.</p

Extended <i>RAS</i> and <i>BRAF</i> Mutation Analysis Using Next-Generation Sequencing

<div><p>Somatic mutations in <i>KRAS</i>, <i>NRAS</i>, and <i>BRAF</i> genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended <i>RAS</i> and <i>BRAF</i> mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within <i>KRAS</i>, <i>NRAS</i>, and <i>BRAF</i>, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10^-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). <i>KRAS</i> mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. <i>KRAS</i> and <i>BRAF</i> mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant <i>RAS</i> and <i>BRAF</i> mutations using FFPE and liquid samples.</p></div

Minimum detection limit of the NGS mutation assay.

<p>Minimum detection limit of the NGS mutation assay.</p

Adjustment of Poisson coefficient as cut-off value.

<p>Results of mutation analysis in 20 FFPE samples when different Poisson coefficients (average ER to average ER plus 7SD) were used. Horizontal axis, base position; vertical axis, 20 FFPE samples from colorectal cancer patients (10 <i>KRAS</i> exon 2 mutation-positive and 10 mutation-negative samples). Blue, specific variant detection at <i>KRAS</i> codons 12 and 13; Red, non-specific variant detection at all positions; Gray, variant detection at unevaluable positions; Yellow, variants other than <i>KRAS</i> codons 12 and 13.</p

Read number and error rate of the NGS assay.

<p>(A) Average read number of 9 amplicons in 10 normal DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon. (B) Error rate of position detection in 10 normal DNA samples (average with standard deviation). Horizontal axis, base position; vertical axis, error rate. (C) Average read number of 9 amplicons in 20 colorectal cancer FFPE DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon.</p