The induction of senescence-like growth arrest by protein kinase C-activating diterpene esters in solid tumor cells.

We previously identified the induction of senescence in melanoma cell lines sensitive to diterpene esters, indicating a therapeutic potential. Here we compared the cytostatic effects of two diterpene esters: the prototypic PKC-activating drug TPA (12-O-tetradecanoylphorbol-13-acetate), and the novel compound PEP008 (20-O-acetyl-ingenol-3-angelate) in cell lines derived from melanoma, breast cancer and colon cancer. The diterpene esters induced permanent growth arrest with characteristics of senescence in a subset of cell lines in all three solid tumor models at 100-1000 ng/ml. Use of the PKC inhibitor bisindolylmaleimide-l demonstrated that activation of PKC was required for growth arrest. Full genome expression profiling identified pivotal genes involved in DNA synthesis and cell cycle control down-regulated by treatment in all three sensitive tumor models. At the protein level, prolonged down-regulation of E2F-1 and proliferating cell nuclear antigen (PCNA), sustained expression of p21(WAF1/CIP1) and dephosphorylation of retinoblastoma (Rb) occurred in the sensitive cells. Additionally, the type II tumor suppressor HRASLS3, which has a role in mitogen-activated protein kinase (MAPK) pathway suppression, was constitutively elevated in cell lines resistant to the senescence effects compared to their sensitive counterparts. Together, these results demonstrate that both TPA and the novel PKC-activating drug PEP008 induce growth arrest with characteristics of senescence in solid tumor cell lines derived from a variety of tissue types, and by a similar mechanism. PKC-activating diterpene esters may therefore have therapeutic potential in a subset of breast cancer, colon cancer and melanoma tumors.

Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation.

BACKGROUND: Thrombospondin-4 (THBS4) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of THBS4 expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter. METHODS: Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of THBS4 transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon. RESULTS: THBS4 expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and THBS4 methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high THBS4 methylation, but the correlation was not significant. THBS4 methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon. CONCLUSIONS: THBS4 shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence THBS4 expression. THBS4 may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation in vitro. THBS4 methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.

Bone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer development.

Bone morphogenic proteins (BMPs) are members of the TGFB growth factor superfamily with well-described functions in bone formation. Although disrupted BMP signalling in tumor development has more recently been investigated, a role for BMP3 in colorectal cancer (CRC) has remained largely unexplored. The aim of this study was to investigate BMP3 disruption in CRCs in relation to both the traditional and serrated pathways of tumor progression. BMP3 was down-regulated as assessed by real-time PCR in 50 of 56 primary tumors (89%). Bisulfite sequencing of the putative promoter revealed extensive hypermethylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aberrant hypermethylation was observed in 33/60 (55%) tumors and was highly correlated with microsatellite instability (P < 0.01), the CpG Island Methylator Phenotype (P < 0.01), BRAF oncogene mutation (P < 0.01), and proximal location (P < 0.001). Methylation was also frequently observed in serrated and traditional adenomatous polyps (22/29, 76%). Re-introduction of BMP3 into cell lines revealed marked growth suppression supporting the functional relevance of this alteration in colorectal tumor development. This study provides molecular and functional data supporting the importance of BMP3 silencing as an early and frequent event in colorectal tumors progressing via the serrated and traditional pathways.

Induction of senescence in diterpene ester-treated melanoma cells via protein kinase C-dependent hyperactivation of the mitogen-activated protein kinase pathway.

The diterpene ester PEP005 is a novel anticancer agent that activates protein kinase C (PKC) and induces cell death in melanoma at high doses. We now describe the in vitro cytostatic effects of PEP005 and the diterpene ester phorbol 12-myristate 13-acetate, observed in 20% of human melanoma cell lines. Primary cultures of normal human neonatal fibroblasts were resistant to growth arrest, indicating a potential for tumor selectivity. Sensitive cell lines were induced to senesce and exhibited a G(1) and G(2)-M arrest. There was sustained expression of p21(WAF1/CIP1), irreversible dephosphorylation of the retinoblastoma protein, and transcriptional silencing of E2F-responsive genes in sensitive cell lines. Activation of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 by PKC was required for diterpene ester-induced senescence. Expression profiling revealed that the MAP kinase inhibitor HREV107 was expressed at a higher transcript level in resistant compared with sensitive cell lines. We propose that activation of PKC overstimulates the RAS/RAF/MEK/ERK pathway, resulting in molecular changes leading to the senescent phenotype.

Tattoo removal with ingenol mebutate.

An increasing number of people are getting tattoos; however, many regret the decision and seek their removal. Lasers are currently the most commonly used method for tattoo removal; however, treatment can be lengthy, costly, and sometimes ineffective, especially for certain colors. Ingenol mebutate is a licensed topical treatment for actinic keratoses. Here, we demonstrate that two applications of 0.1% ingenol mebutate can efficiently and consistently remove 2-week-old tattoos from SKH/hr hairless mice. Treatment was associated with relocation of tattoo microspheres from the dermis into the posttreatment eschar. The skin lesion resolved about 20 days after treatment initiation, with some cicatrix formation evident. The implications for using ingenol mebutate for tattoo removal in humans are discussed.

SnoN expression is differently regulated in microsatellite unstable compared with microsatellite stable colorectal cancers.

BACKGROUND: SnoN is an important regulator of the transforming growth factor beta (TGFbeta) signalling pathway and has been shown to exhibit both tumour promotion and suppression activity. METHODS: To further explore the role of this complex molecule in colorectal tumorigenesis, we examined 52 paired normal and tumour colorectal specimens stratified by level of microsatellite instability; 18 with high-level microsatellite instability (MSI-H) and 34 microsatellite stable (MSS). SnoN transcript expression was quantitated by real-time PCR and analysed with respect to clinical indicators of prognosis. RESULTS: Within the MSI-H subgroup, SnoN was commonly either up-regulated (6/18, 33%) or down-regulated (7/18, 39%). A significantly different distribution of SnoN expression was observed in MSS cancers compared with MSI-H (P < or = 0.001). Whilst 17/34 (50%) of MSS tumours demonstrated up-regulation, none showed down-regulated expression. Within the MSI-H subgroup, up-regulation was significantly correlated with lack of repeat tract mutation in the TGFbetaRII gene (P < or = 0.025), suggesting that SnoN is more frequently up-regulated in the presence of functional TGFbeta signalling. CONCLUSION: Together these data support the notion that SnoN has both oncogenic and tumour suppressive properties depending on other genetic changes within the tumour, and that the MSI-H pathway of colorectal tumorigenesis presents an excellent model for the study of these opposing functions.

Antitumor activity of 3-ingenyl angelate: plasma membrane and mitochondrial disruption and necrotic cell death.

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodessication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 micro g) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; n = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; n = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Foxn1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD(90) for PEP005 for a panel of tumor cell lines was 180-220 micro M. Electron microscopy showed that treatment with PEP005 both in vitro (230 micro M) and in vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. (51)Cr release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 micro M) treatment of tumor cells in vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Microarray expression profiling in melanoma reveals a BRAF mutation signature.

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.

Effective treatment of squamous cell carcinomas with ingenol mebutate gel in immunologically intact SKH1 mice.

Ingenol mebutate has recently been approved by the Federal Drug Administration (USA) as a topical treatment for actinic keratoses. Herein, we describe the efficacy of ingenol mebutate for the topical treatment of squamous cell carcinoma (SCC) using a wild-type mouse model (SKH1) and the UV-induced mouse SCC cell line, T7. Daily treatment for 2 days with 0.25 % ingenol mebutate gel produced a cure rate of 70 %, with 0 % for placebo gel. Electron microscopy revealed swelling of cancer cell mitochondria within 1 h, with disruption of the inner mitochondrial membranes evident at 6 h post treatment. Primary necrosis of cancer cells was clearly evident by 24 h. Treatment was associated with local haemorrhage and a prodigious neutrophil infiltrate, with anti-T7 antibodies also detected. This is the first report of the successful treatment of SCC tumours with ingenol mebutate gel in wild-type mice, and supports the view that ingenol mebutate induces primary necrosis and activates the immune system.

Ingenol mebutate field-directed treatment of UVB-damaged skin reduces lesion formation and removes mutant p53 patches.

Skin cancer is the most prevalent cancer worldwide and is primarily caused by chronic UV exposure. Here, we describe the topical field-directed treatment of SKH1/hr mice with UVB-damaged skin with ingenol mebutate, a new topical drug shown to be effective for the treatment of actinic keratosis (AK). Application of 0.05% ingenol mebutate gel to photo-damaged skin resulted in a ≈70% reduction in the number of skin lesions that subsequently emerged compared with placebo treatment. Ingenol mebutate treatment also reduced the number of mutant p53 keratinocyte patches by ≈70%. The treatment resulted in epidermal cell death, acute inflammation, recruitment of neutrophils, hemorrhage, and eschar formation, all of which resolved over several weeks. Ingenol mebutate field-directed treatment might thus find utility in the removal of subclinical precancerous cells from UV-damaged skin. Field-directed treatment may be particularly suitable for patients who have AKs surrounded by UV-damaged skin.