High Expression of Wee1 Is Associated with Poor Disease-Free Survival in Malignant Melanoma: Potential for Targeted Therapy

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents

The fatty acid binding protein 7 (FABP7) is involved in proliferation and invasion of melanoma cells

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms underlying melanoma tumor development and progression are still not completely understood. One of the new candidates that emerged from a recent gene expression profiling study is <it>fatty acid-binding protein 7 </it>(<it>FABP7)</it>, involved in lipid metabolism, gene regulation, cell growth and differentiation.</p> <p>Methods</p> <p>We studied the functional role of FABP7 in human melanoma cell lines and using immunohistochemistry analyzed its expression pattern and clinical role in 11 nevi, 149 primary melanomas and 68 metastases.</p> <p>Results</p> <p>FABP7 mRNA and protein level is down-regulated following treatment of melanoma cell lines with a PKC activator (PMA) or MEK1 inhibitor (PD98059). Down-regulation of FABP7 using siRNA decreased cell proliferation and invasion but did not affect apoptosis. In clinical specimens, FABP7 was expressed in 91% of nevi, 71% of primary melanomas and 70% of metastases, with a cytoplasmic and/or nuclear localization. FABP7 expression was associated with tumor thickness in superficial spreading melanoma (P = 0.021). In addition, we observed a trend for an association between FABP7 expression and Ki-67 score (P = 0.070) and shorter relapse-free survival (P = 0.069) in this group of patients.</p> <p>Conclusion</p> <p>Our data suggest that FABP7 can be regulated by PKC and the MAPK/ERK1/2 pathway through independent mechanisms in melanoma cell lines. Furthermore, FABP7 is involved in cell proliferation and invasion <it>in vitro</it>, and may be associated with tumor progression in melanoma.</p

The magnitude of translational and rotational head accelerations experienced by riders during downhill mountain biking

Objectives To determine the magnitude of translational and rotational head accelerations during downhill mountain biking. Design Observational study Methods Sixteen male downhill cyclists (age 26.4 ± 8.4 years; stature 179.4 ± 7.2 cm; mass 75.3 ± 5.9 kg) were monitored during two rounds of the British Downhill Series. Riders performed two runs on each course wearing a triaxial accelerometer behind the right ear. The means of the two runs for each course were used to determine differences between courses for mean and maximum peak translational (g) and rotational accelerations (rads/s2) and impact duration for each course. Results Significant differences (p 10 g), FW = 12.5 ± 7.6, RYF = 42.8 ± 27.4 (t(22.96) = -4.70; p < 0.001; 95 % CI = 17.00 to 43.64); maximum peak rotational acceleration, FW = 6805.4 ± 3073.8 rads/s2, RYF = 9799.9 ± 3381.7 rads/s2 (t(32) = -2.636; p = 0.01; 95 % CI = 680.31 to 5308.38); mean acceleration duration FW = 4.7 ± 1.2 ms, RYF = 6.5 ± 1.4 ms (t(32) = -4.05; p < 0.001; 95 % CI = 0.91 to 2.76) and maximum acceleration duration, FW = 11.6 ± 4.5 ms, RYF = 21.2 ± 9.1 (t(29.51) = -4.06; p = 0.001; 95 % CI = 4.21 to 14.94). No other significant differences were found. Conclusions Findings indicate that downhill riders may be at risk of sustaining traumatic brain injuries and course design influences the number and magnitude of accelerations

The increased expression of fatty acid-binding protein 9 in prostate cancer and its prognostic significance

In contrast to numerous studies conducted to investigate the crucial role of fatty acid binding protein 5 (FABP5) in prostate cancer, investigations on the possible involvement of other FABPs are rare. Here we first measured the mRNA levels of 10 FABPs in benign and malignant prostate cell lines and identified the differentially expressed FABP6 and FABP9 mRNAs whose levels in all malignant cell lines were higher than those in the benign cells. Thereafter we assessed the expression status of FABP6 and FABP9 in both prostate cell lines and in human tissues. FABP6 protein was overexpressed only in 1 of the 5 malignant cell lines and its immunostaining intensities were not significantly different between benign and malignant prostate tissues. In contrast, FABP9 protein was highly expressed in highly malignant cell lines PC-3 and PC3-M, but its level in the benign PNT-2 and other malignant cell lines was not detectable. When analysed in an archival set of human prostate tissues, immunohistochemical staining intensity for FABP9 was significantly higher in carcinomas than in benign cases and the increase in FABP9 was significantly correlated with reduced patient survival times. Moreover, the increased level of staining for FABP9 was significantly associated with the increased joint Gleason scores (GS) and androgen receptor index (AR). Suppression of FABP9 expression in highly malignant PC3-M cells inhibited their invasive potential. Our results suggest that FABP9 is a valuable prognostic marker to predict the outcomes of prostate cancer patients, perhaps by playing an important role in prostate cancer cell invasion

Increased expression of h-prune is associated with tumor progression and poor survival in gastric cancer

The human homolog of the Drosophila prune protein (from PRUNE, which encodes h-prune), which interacts with glycogen synthase kinase 3, promotes cellular motility. H-prune also interacts with nm23-H1, a suppressor of cancer metastasis. It has been reported that stimulation of cellular motility by h-prune is enhanced by its interaction with nm23-H1 in breast cancer cells. In the present study, we examined the expression of h-prune and nm23-H1 during tumor progression in gastric cancer (GC). PRUNE mRNA was overexpressed in 12 (32%) of 38 GC cases by quantitative reverse transcription-polymerase chain reaction. PRUNE mRNA levels correlated significantly with advanced T grade, N grade and tumor stage. Immunohistochemical analysis revealed that 43 (30%) of 143 GC cases were positive for h-prune, and h-prune-positive GC cases showed more advanced T grade, N grade and tumor stage than h-prune-negative GC cases. One hundred and twenty-four (87%) of 143 GC cases were positive for nm23-H1, and nm23-H1 was expressed in almost all (42 cases, 98%) h-prune-positive GC cases. Many GC cases positive for both h-prune and nm23-H1 showed more advanced T grade, N grade and tumor stage than other type GC cases. Patients with h-prune-positive GC had a significantly worse survival rate than patients with h-prune-negative GC. These findings indicate that overexpression of h-prune is associated with tumor progression and aggressiveness of GC. nm23-H1 may enhance motility of cancer cells by interacting with h-prune. (Cancer Sci 2007; 98: 1198-1205

Serologic and immunohistochemical prognostic biomarkers of cutaneous malignancies

Biomarkers are important tools in clinical diagnosis and prognostic classification of various cutaneous malignancies. Besides clinical and histopathological aspects (e.g. anatomic site and type of the primary tumour, tumour size and invasion depth, ulceration, vascular invasion), an increasing variety of molecular markers have been identified, providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities, up to even changing existing classification systems. Recently published gene expression or proteomic profiling data relate to new marker molecules involved in skin cancer pathogenesis, which may, after validation by suitable studies, represent future prognostic or predictive biomarkers in cutaneous malignancies. We, here, give an overview on currently known serologic and newer immunohistochemical biomarker molecules in the most common cutaneous malignancies, malignant melanoma, squamous cell carcinoma and cutaneous lymphoma, particularly emphasizing their prognostic and predictive significance

A Context-Specific Role for Retinoblastoma Protein-Dependent Negative Growth Control in Suppressing Mammary Tumorigenesis

The ability to respond to anti-growth signals is critical to maintain tissue homeostasis and loss of this negative growth control safeguard is considered a hallmark of cancer. Negative growth regulation generally occurs during the G0/G1 phase of the cell cycle, yet the redundancy and complexity among components of this regulatory network has made it difficult to discern how negative growth cues protect cells from aberrant proliferation.The retinoblastoma protein (pRB) acts as the final barrier to prevent cells from entering into the cell cycle. By introducing subtle changes in the endogenous mouse Rb1 gene (Rb1(ΔL)), we have previously shown that interactions at the LXCXE binding cleft are necessary for the proper response to anti-growth signals such as DNA damage and TGF-β, with minimal effects on overall development. This disrupts the balance of pro- and anti-growth signals in mammary epithelium of Rb1(ΔL/ΔL) mice. Here we show that Rb1(ΔL/ΔL) mice are more prone to mammary tumors in the Wap-p53(R172H) transgenic background indicating that negative growth regulation is important for tumor suppression in these mice. In contrast, the same defect in anti-growth control has no impact on Neu-induced mammary tumorigenesis.Our work demonstrates that negative growth control by pRB acts as a crucial barrier against oncogenic transformation. Strikingly, our data also reveals that this tumor suppressive effect is context-dependent

p53 mutations in human cutaneous melanoma correlate with sun exposure but are not always involved in melanomagenesis

In melanoma, the relationship between sun exposure and the origin of mutations in either the N-ras oncogene or the p53 tumour-suppressor gene is not as clear as in other types of skin cancer. We have previously shown that mutations in the N-ras gene occur more frequently in melanomas originating from sun-exposed body sites, indicating that these mutations are UV induced. To investigate whether sun exposure also affects p53 in melanoma, we analysed 81 melanoma specimens for mutations in the p53 gene. The mutation frequency is higher than thus far reported: 17 specimens (21%) harbour one or more p53 mutations. Strikingly, 17 out of 22 mutations in p53 are of the C:G to T:A or CC:GG to TT:AA transitional type, strongly suggesting an aetiology involving UV exposure. Interestingly, the p53 mutation frequency in metastases was much lower than in primary tumours. In the case of metastases, a role for sun exposure was indicated by the finding that the mutations are present exclusively in skin metastases and not in internal metastases. Together with a relatively frequent occurrence of silent third-base pair mutations in primary melanomas, this indicates that the p53 mutations, at least in these tumours, have not contributed to melanomagenesis and may have originated after establishment of the primary tumour. 1999 Cancer Research Campaig

MDM2 antagonist Nutlin-3a potentiates antitumour activity of cytotoxic drugs in sarcoma cell lines

<p>Abstract</p> <p>Background</p> <p>Frequent failure and severe side effects of current sarcoma therapy warrants new therapeutic approaches. The small-molecule MDM2 antagonist Nutlin-3a activates the p53 pathway and efficiently induces apoptosis in tumours with amplified <it>MDM2 </it>gene and overexpression of MDM2 protein. However, the majority of human sarcomas have normal level of MDM2 and the therapeutic potential of MDM2 antagonists in this group is still unclear. We have investigated if Nutlin-3a could be employed to augment the response to traditional therapy and/or reduce the genotoxic burden of chemotherapy.</p> <p>Methods</p> <p>A panel of sarcoma cell lines with different <it>TP53 </it>and <it>MDM2 </it>status were treated with Nutlin-3a combined with Doxorubicin, Methotrexate or Cisplatin, and their combination index determined.</p> <p>Results</p> <p>Clear synergism was observed when Doxorubicin and Nutlin-3a were combined in cell lines with wild-type <it>TP53 </it>and amplified <it>MDM2</it>, or with Methotrexate in both <it>MDM2 </it>normal and amplified sarcoma cell lines, allowing for up to tenfold reduction of cytotoxic drug dose. Interestingly, Nutlin-3a seemed to potentiate the effect of classical drugs as Doxorubicin and Cisplatin in cell lines with mutated <it>TP53</it>, but inhibited the effect of Methotrexate.</p> <p>Conclusion</p> <p>The use of Nutlin in combination with classical sarcoma chemotherapy shows promising preclinical potential, but since clear biomarkers are still lacking, clinical trials should be followed up with detailed tumour profiling.</p

Epigenetics of human cutaneous melanoma: setting the stage for new therapeutic strategies

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects