Temozolomide induces senescence but not apoptosis in human melanoma cells

Temozolomide (TMZ), a DNA alkylating agent used in the treatment of melanoma, is believed to mediate its effect by addition of a methyl group to the O6 position of guanine in DNA. Resistance to the agent may be in part due to the activity of O6-methylguanine-DNA methyl transferase (MGMT). In the present study, we show that sensitivity of melanoma cells to TMZ was dependent on their p53 status and levels of MGMT. Analysis of the mechanisms underlying reduced viability showed no evidence for induction of apoptosis even though marked levels of apoptosis was seen in TK6 lymphoma cells. Sensitivity of melanoma cells was associated with p53-dependent G2/M cell cycle arrest and induction of senescence. To verify the role of p53, the assays were repeated in presence of pifithrin-α, an inhibitor of p53. This resulted in increased viability of melanoma cells with wild-type p53 and reversed G2/M cell cycle arrest. Paradoxically, apoptosis was increased in melanoma but decreased as expected in TK6 lymphoma cells. These results are consistent with the view that TMZ is relatively ineffective against melanoma due to defective apoptotic signalling resulting from activation of p53. The nature of the defects in apoptotic signalling remains to be explored

Dendritic cell density and activation status in human breast cancer – CD1a, CMRF-44, CMRF-56 and CD-83 expression

Low CD1a-positive putative dendritic cell numbers in human breast cancer has recently been described and may explain the apparent ‘poor immunogenicity’ previously reported in breast cancer. Little attention has been given to dendritic cell activation within the tumour microenvironment, which is another reason why the in-situ immune response may be severely deficient. We have therefore examined CD1a expression as a marker for dendritic cells, together with CMRF-44 and -56 as markers of dendritic cell activation status, in 40 human breast cancers. The results demonstrate few or no CD1a-positive putative dendritic cells and minimal or no expression of the dendritic cell activation markers. Both dendritic cell number and dendritic cell activation appear substantially deficient in human breast cancers, regardless of tumour histological grade

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe

Macrophage migration inhibitory factor engages PI3K/Akt signalling and is a prognostic factor in metastatic melanoma

Background: Macrophage migration inhibitory factor (MIF) is a widely expressed cytokine involved in a variety of cellular processes including cell cycle regulation and the control of proliferation. Overexpression of MIF has been reported in a number of cancer types and it has previously been shown that MIF is upregulated in melanocytic tumours with the highest expression levels occurring in malignant melanoma. However, the clinical significance of high MIF expression in melanoma has not been reported. Methods: MIF expression was depleted in human melanoma cell lines using siRNA-mediated gene knockdown and effects monitored using in vitro assays of proliferation, cell cycle, apoptosis, clonogenicity and Akt signalling. In silico analyses of expression microarray data were used to correlate MIF expression levels in melanoma tumours with overall patient survival using a univariate Cox regression model. Results: Knockdown of MIF significantly decreased proliferation, increased apoptosis and decreased anchorage-independent growth. Effects were associated with reduced numbers of cells entering S phase concomitant with decreased cyclin D1 and CDK4 expression, increased p27 expression and decreased Akt phosphorylation. Analysis of clinical outcome data showed that MIF expression levels in primary melanoma were not associated with outcome (HR = 1.091, p = 0.892) whereas higher levels of MIF in metastatic lesions were significantly associated with faster disease progression (HR = 2.946, p = 0.003 and HR = 4.600, p = 0.004, respectively in two independent studies). Conclusions: Our in vitro analyses show that MIF functions upstream of the PI3K/Akt pathway in human melanoma cell lines. Moreover, depletion of MIF inhibited melanoma proliferation, viability and clonogenic capacity. Clinically, high MIF levels in metastatic melanoma were found to be associated with faster disease recurrence. These findings support the clinical significance of MIF signalling in melanoma and provide a strong rationale for both targeting and monitoring MIF expression in clinical melanoma

TRAIL-induced apoptosis of human melanoma cells involves activation of caspase-4

Although it is conventionally regarded as an inflammatory caspase, recent studies have shown that caspase-4 plays a role in induction of apoptosis by endoplasmic reticulum (ER) stress. We report here that activation of caspase-4 is also involved in induction of apoptosis by TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells. Treatment with TRAIL resulted in activation of caspase-4. This appeared to be mediated by caspase-3, in that caspase-4 was activated later than caspase-8, -9, and -3, and that inhibition of caspase-3 blocked TRAIL-induced caspase-4 activation. Notably, TRAIL triggered ER stress in melanoma cells as shown by up-regulation of the GRP78 protein and the spliced form of XBP-1 mRNA. This seemed to be necessary for activation of caspase-4, as activation of caspase-3 by agents that did not trigger ER stress did not cause activation of caspase-4. Importantly, inhibition of caspase-4 also partially blocked caspase-3 activation, suggesting that activation of caspase-4 may be positive feed-back mechanism to further enhance caspase-3 activation. Collectively, these results show that activation of caspase-4 contributes to TRAIL-induced apoptosis and is associated with induction of ER stress by TRAIL in melanoma cells, and may have important implications for improving therapeutic efficacies of TRAIL in melanoma

Involvement of endoplasmic reticulum stress in docetaxel-induced JNK-dependent apoptosis of human melanoma

Our previous studies revealed that Docetaxelinduced apoptosis of melanoma cells is entirely dependent on activation of the JNK signalling pathway. Here, we show that Docetaxel-induced apoptosis is mediated by induction of ER stress. This was shown by Docetaxelinduced activation of proteins involved in ER stress signalling namely GRP78, ATF6, IRE1α, and PERK/eIF2α. Knockdown of IRE1α by siRNA markedly inhibited Docetaxel-induced JNK activation and downstream targets of JNK indicating that activation of IRE1α was upstream of activation of the JNK. Co-immunoprecipitation experiments showed that activation of JNK is due to activation of ASK1 through formation of an IRE1α-TRAF2-ASK1 complex. ER stress mediated activation of the JNK pathway is downstream of activation of PKCδ in that downregulation of PKCδ expression using specific PKCδ siRNA significantly inhibited Docetaxel-induced activation of IRE1α and the JNK pathway. These findings provide new insights to understand the mode of action of taxanes in treatment of human melanoma