Ana Slipicevic

Expression of Activated Akt and PTEN in Malignant Melanomas Relationship With Clinical Outcome

Our purpose was to analyze, by immunohisto-chemistry, the expression of activated serine-threonine protein kinase B (p-Akt) and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in benign nevi and primary and metastatic melanomas and to correlate the expression level with clinical variables. We observed cytoplasmic and/or nuclear expression of p-Akt in 22 (54%) of 41 benign nevi, 112 (71.3%) of 157 primary tumors, and 50 (71%) of 70 metastases. Cytoplasmic PTEN staining was observed in 0 (0%), 152 (87.7%), and 64 (90%) of 41 nevi, 162 primary tumors, and 71 metastases, respectively. A significant positive correlation was seen between PTEN and p-Akt cytoplasmic expression (P < .001) in primary melanomas. Cytoplasmic p-Akt expression showed a positive association with cyclin A in superficial spreading (P = .038) but not in nodular (P = .22) melanomas. Cytoplasmic p-Akt and PTEN expression did not have an impact on disease-free and overall survival, but complete lack of nuclear p-Akt expression was a predictor of shorter disease-free survival (P = .025) for patients with superficial spreading melanoma.

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

BackgroundThe 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 fatty acid-binding protein 7 (FABP7), involved in lipid metabolism, gene regulation, cell growth and differentiation.MethodsWe 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.ResultsFABP7 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.ConclusionOur 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 in vitro, and may be associated with tumor progression in melanoma.

Wnt5A promotes an adaptive, senescent‐like stress response, while continuing to drive invasion in melanoma cells

We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAFV600E mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence‐associated β‐galactosidase (SA‐β‐gal), senescence‐associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA‐β‐gal and SAHF, these Wnt5A‐high cells are able to colonize the lungs in in vivo tail vein colony‐forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.

Cytoplasmic BRMS1 expression in malignant melanoma is associated with increased disease-free survival

Background/aimsBreast cancer metastasis suppressor 1 (BRMS1) blocks metastasis in melanoma xenografts; however, its usefulness as a biomarker in human melanomas has not been widely studied. The goal was to measure BRMS1 expression in benign nevi, primary and metastatic melanomas and evaluate its impact on disease progression and prognosis.MethodsParaffin-embedded tissue from 155 primary melanomas, 69 metastases and 15 nevi was examined for BRMS1 expression using immunohistochemistry. siRNA mediated BRMS1 down-regulation was used to study impact on invasion and migration in melanoma cell lines.ResultsA significantly higher percentage of nevi (87%), compared to primary melanomas (20%) and metastases (48%), expressed BRMS1 in the nucelus (p < 0.0001). Strong nuclear staining intensity was observed in 67% of nevi, and in 9% and 24% of the primary and metastatic melanomas, respectively (p < 0.0001). Comparable cytoplasmic expression was observed (nevi; 87%, primaries; 86%, metastases; 72%). However, a decline in cytoplasmic staining intensity was observed in metastases compared to nevi and primary tumors (26%, 47%, and 58%, respectively, p < 0.0001). Score index (percentage immunopositive celles multiplied with staining intensity) revealed that high cytoplasmic score index (≥ 4) was associated with thinner tumors (p = 0.04), lack of ulceration (p = 0.02) and increased disease-free survival (p = 0.036). When intensity and percentage BRMS1 positive cells were analyzed separately, intensity remained associated with tumor thickness (p = 0.024) and ulceration (p = 0.004) but was inversely associated with expression of proliferation markers (cyclin D3 (p = 0.008), cyclin A (p = 0.007), and p21Waf1/Cip1 (p = 0.009)). Cytoplasmic score index was inversely associated with nuclear p-Akt (p = 0.013) and positively associated with cytoplasmic p-ERK1/2 expression (p = 0.033). Nuclear BRMS1 expression in ≥ 10% of primary melanoma cells was associated with thicker tumors (p = 0.016) and decreased relapse-free period (p = 0.043). Nuclear BRMS1 was associated with expression of fatty acid binding protein 7 (FABP7; p = 0.011), a marker of invasion in melanomas. In line with this, repression of BRMS1 expression reduced the ability of melanoma cells to migrate and invade in vitro.ConclusionOur data suggest that BRMS1 is localized in cytoplasm and nucleus of melanocytic cells and that cellular localization determines its in vivo effect. We hypothesize that cytoplasmic BRMS1 restricts melanoma progression while nuclear BRMS1 possibly promotes melanoma cell invasion.Please see related article: http://www.biomedcentral.com/1741-7015/10/19

Combined inhibition of the cell cycle related proteins Wee1 and Chk1/2 induces synergistic anti-cancer effect in melanoma

BackgroundMalignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy. Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression. This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets.MethodsIn the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models.ResultsOur in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X) - and premature mitosis of S-phase cells. Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts.ConclusionsThese data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.

Wee1 is a novel independent prognostic marker of poor survival in post-chemotherapy ovarian carcinoma effusions.

OBJECTIVE Wee1-like kinase (Wee1) is a tyrosine kinase which negatively regulates entry into mitosis at the G2 to M-phase transition and has a role in inhibition of unscheduled DNA replication in S-phase. The present study investigated the clinical role of Wee1 in advanced-stage (FIGO III-IV) ovarian serous carcinoma. METHODS Wee1 protein expression was analyzed in 287 effusions using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including survival. Forty-five effusions were additionally studied using Western blotting. Wee1 was further silenced in SKOV3 and OVCAR8 cells by siRNA knockdown and proliferation was assessed. RESULTS Nuclear expression of Wee1 in tumor cells was observed in 265/287 (92%) and 45/45 (100%) effusions by immunohistochemistry and Western blotting, respectively. Wee1 expression by immunohistochemistry was significantly higher in post-chemotherapy disease recurrence compared to pre-chemotherapy effusions obtained at diagnosis (p=0.002). Wee1 silencing in SKOV3 and OVCAR8 cells reduced proliferation. In univariate survival analysis of the entire cohort, a trend was observed between high (>25% of cells) Wee1 expression and poor overall survival (p=0.083). Survival analysis for 109 patients with post-chemotherapy effusions showed significant association between Wee1 expression and poor overall survival (p=0.004), a finding which retained its independent prognostic role in Cox multivariate analysis (p=0.003). CONCLUSIONS Wee1 is frequently expressed in ovarian serous carcinoma effusions, and its expression is significantly higher following exposure to chemotherapy. The present study is the first to report that Wee1 is an independent prognostic marker in serous ovarian carcinoma.

Biological effects induced by insulin‐like growth factor binding protein 3 (IGFBP‐3) in malignant melanoma

The insulin like growth factor (IGF) signaling pathway has been shown to contribute to melanoma progression, but little is known about the role of the IGF binding protein 3 (IGFBP‐3) in melanoma biology. The aim of the present study was to characterize expression, function and regulation of IGFBP‐3 in malignant melanomas and study its potential as a biomarker. The expression of IGFBP‐3 varied between different human melanoma cell lines and reintroduction of the protein in non‐expressing cells led to induction of apoptosis. Interestingly, in cell lines expressing endogenous IGFBP‐3, siRNA silencing of the protein led to a cell line‐dependent decrease in proliferation, but had no effect on apoptosis and invasion. Examination of patient material showed that IGFBP‐3 is unexpressed in benign nevi while a slight increase in protein expression was seen in primary and metastatic melanoma. However, expression of the protein was low and no correlation was found with circulating levels of IGFBP‐3 in serum, suggesting that IGFBP‐3 has limited potential as a predictive marker in malignant melanoma. We showed that promoter methylation of IGFBP‐3 occurred in both melanoma cell lines and patient material, implicating epigenetic silencing as a regulation mechanism. Furthermore, expression of the protein was shown to be regulated by the PI3‐kinase/AKT and MAPK/ERK1/2 pathways. In summary, our findings suggest that IGFBP‐3 can exert dual functional effects influencing both apoptosis and proliferation. Development of resistance to the antiproliferative effects of IGFBP‐3 may be an important step in progression of malignant melanomas.

Diagnostic and prognostic role of the insulin growth factor pathway members insulin-like growth factor-II and insulin-like growth factor binding protein-3 in serous effusions

We recently reported on higher expression of the insulin-like growth factor pathway genes IGF-II and IGFBP3 in serous ovarian/peritoneal carcinoma compared to malignant peritoneal mesothelioma. The present study analyzed the diagnostic and clinical role of these proteins in serous effusions. Effusions (n = 327), including 294 carcinomas (205 ovarian, 48 breast, 17 cervical/endometrial, 12 lung, 12 gastrointestinal/genitourinary) and 33 malignant mesotheliomas, were immunostained for insulin-like growth factor-II and insulin-like growth factor binding protein-3. Surgical ovarian carcinoma (n = 124) and peritoneal mesothelioma (n = 18) specimens were additionally studied. Insulin-like growth factor binding protein-3 levels were measured in 148 effusion supernatants (114 ovarian carcinomas, 18 breast carcinomas, 16 mesotheliomas) using enzyme-linked immunosorbent assay. Insulin-like growth factor binding protein-3 promoter methylation was analyzed in 11 ovarian carcinoma effusions. Insulin-like growth factor binding protein-3 (P = .002) and insulin-like growth factor-II (P < .001) expression by immunohistochemistry was significantly higher in carcinomas compared to mesotheliomas, with diagnostic sensitivity of 77% and 70% and specificity of 55% and 70%, respectively. In surgical specimens, insulin-like growth factor binding protein-3 expression was higher in ovarian carcinomas compared to peritoneal mesotheliomas (P = .007), whereas insulin-like growth factor-II expression was comparable (P = .505). Insulin-like growth factor binding protein-3 levels by enzyme-linked immunosorbent assay were comparable in the 3 analyzed cancer types. Insulin-like growth factor binding protein-3 promoter methylation was found in 6 of 11 effusions. High insulin-like growth factor binding protein-3 expression in prechemotherapy and high insulin-like growth factor-II expression in postchemotherapy ovarian carcinoma effusions correlated with poor overall survival (P = .031 and P = .024, respectively). Insulin-like growth factor-II expression in postchemotherapy effusions was an independent prognostic factor in Cox multivariate analysis (P = .04). In conclusion, insulin-like growth factor-II and insulin-like growth factor binding protein-3 are more frequently expressed in metastatic carcinomas compared to mesothelioma in effusions but are less specific than currently used markers. Insulin-like growth factor-II and insulin-like growth factor binding protein-3 may be novel prognostic markers in metastatic ovarian carcinoma.

KIT in Melanoma: Many Shades of Gray

Activating mutations in KIT have been identified in melanomas of acral and mucosal types and in those arising in chronically sun-damaged skin. Until now, KIT has been considered an oncogenic driver and a potential therapeutic target. However, data presented by Dhal et al. show that in cutaneous melanomas the KIT promoter is a target for hypermethylation, leading to its downregulation. Their observations suggest that signaling pathways downstream of KIT may have distinct and opposing roles in the pathogenesis of melanoma subtypes. This will have important implications for the use of KIT inhibitors in treating melanomas.