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

Transfection with siWee1 promotes DNA damages and apoptosis.

<p>A. Presence of cytoplasmic oligonucleosomes was measured by ELISA following 48 h siWee1 transfection. Induction of apoptosis shown as enrichment factor calculated as absorbance at 405 nm of siWee1 treated cells relative to siCtr treated cells. B. Protein expressions were measured by Western blot following 48 h transfection with either siCtr or siWee1. Cleavage of PARP and Caspase 3 are shown with arrows. α-tubulin was used as loading control. The figure is representative of at least three independent biological experiments.</p

Wee1 expression correlates with clinical parameters- and markers of tumor progression.

*<p>Low expression of Cyclin A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes1" target="_blank">[11]</a>, Ki67 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes1" target="_blank">[11]</a>, Cyclin D3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes2" target="_blank">[12]</a>, p21 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes3" target="_blank">[13]</a> and p53 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Maelandsmo1" target="_blank">[14]</a>; defined as immunoreactivity in <5% of the tumor cells. Wee1 expression in <10% of tumor cells is defined as low.</p>†<p>Statistical significances determined by Chi-square tests.</p

Transfection with siWee1 leads to alterations in cell cycle distribution (A) and -associated proteins (B).

<p>A. Histograms showing cell cycle distribution after siWee1 transfection for 48 hours, measured by flow cytometry. B. Cells were treated with siWee1 for 48 hours and protein expressions were analyzed by immunoblotting using the indicated antibodies. α-tubulin was used as loading control. The figure is representative of at least three independent biological experiments.</p

High Wee1 expression increases with tumor progression and is associated with a shorter relapse-free period.

<p>A. Wee1 expression in cultured melanocytes, benign nevi, primary- and metastatic melanoma, analyzed by immunohistochemistry. B. Melanoma patients were grouped according to Wee1 expression in their tumors (high (n = 44) or low (n = 63)). Relapse-free survival in months was estimated for both groups and presented as a Kaplan Meyer curve.</p

Transfection with siWee1 effectively shuts down Wee1 expression and reduces cell viability.

<p>A. Cells were transfected with siWee1 for 48 hours. Expressions of Wee1 and pCDK1^Tyr15 were examined by western blot analysis. α-tubulin was used as loading control. The figure is representative of at least three independent biological experiments.B and C. Cells were transfected with siWee1 (dots: 24 h, stripes: 48 h and no-pattern: 72 h). The relative amount of viable cells was estimated by MTS (B), and the relative quantity of living cells was estimated by counting trypan-excluding cells (C).</p

Number (percentage) of melanocytic lesions expressing different levels of Wee1.

<p>Wee1 expressions in benign nevi, primary- and metastatic melanoma were estimated by immunohistochemistry, and categorized in four semi-quantitative classes according to percentage of immunoreactive tumor cells. The groups were further divided into low (<10%) and high (≥10%) expression.</p

A Three-dimensional Ex Vivo Viability Assay Reveals a Strong Correlation Between Response to Targeted Inhibitors and Mutation Status in Melanoma Lymph Node Metastases

Although clinical management of melanoma has changed considerably in recent years, intrinsic treatment resistance remains a severe problem and strategies to design personal treatment regimens are highly warranted. We have applied a three-dimensional (3D) ex vivo drug efficacy assay, exposing disaggregated cells from 38 freshly harvested melanoma lymph node metastases and 21 patient derived xenografts (PDXs) to clinical relevant drugs for 7 days, and examined its potential to evaluate therapy response. A strong association between Vemurafenib response and BRAF mutation status was achieved (P ex vivo results, two tumors diagnosed as BRAF wild-type by routine pathology examinations had to be re-evaluated; one was subsequently found to have a complex V600E mutation, the other a double BRAF mutation (V600E/K601 N). No BRAF inhibitor resistance mechanisms were identified, but PIK3CA and NF1 mutations were identified in two highly responsive tumors. Concordance between ex vivo drug responses using tissue from PDXs and corresponding patient tumors demonstrate that PDX models represent an indefinite source of tumor material that may allow ex vivo evaluation of numerous drugs and combinations, as well as studies of underlying molecular mechanisms. In conclusion, we have established a rapid and low cost ex vivo drug efficacy assay applicable on tumor tissue from patient biopsies. The 3D/spheroid format, limiting the influence from normal adjacent cells and allowing assessment of drug sensitivity to numerous drugs in one week, confirms its potential as a supplement to guide clinical decision, in particular in identifying non-responding patients