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

BACKGROUND: Malignant 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. METHODS: In 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. RESULTS: Our 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. CONCLUSIONS: These data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.

High expression of wee1 is associated with malignancy in vulvar squamous cell carcinoma patients.

BACKGROUND: Vulvar squamous cell carcinoma is a cancer form with increasing incidence rate and few treatment options. Wee1 is a central regulator of the G2/M DNA-damage checkpoint, and has in previous studies been described as a prognostic biomarker and a potential target for therapy in other cancer forms. METHODS: In the present study we analyzed the expression of Wee1 in a panel of 297 vulvar tumors by immunohistochemistry. Furthermore, siRNA transfections were carried out in two vulvar cancer cell lines (SW-954 and CAL-39) in order to study the effect on cell cycle distribution (flow cytometry) and proteins (western blot) involved in DNA damage response and apoptosis. RESULTS: Wee1 kinase is increased in vulvar squamous cell carcinomas, as compared to expression in normal epithelium, and a high Wee1 expression is associated with markers of malignancy, such as lymph node metastasis and poor differentiation. Our in vitro results showed that siRNA mediated Wee1 silencing only led to a modest reduction in viability, when examined in vulvar cancer cell lines. Nonetheless, a marked increase in DNA damages, as assessed by augmented levels of γ-H2AX, was observed in both cell lines in the absence of Wee1. CONCLUSIONS: Our results suggest that Wee1 may be involved in the progression of vulvar carcinomas. Based on our in vitro results, Wee1 is unlikely to function as a target for mono-treatment of these patients.

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 G(2)/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), p21(Cip1/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; WM239(WTp53), WM45.1(MUTp53) and LOX(WTp53), 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 G(1)/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.

Synthetic retinoid CD437 induces apoptosis and acts synergistically with TRAIL receptor-2 agonist in malignant melanoma.

The novel synthetic retinoid, CD437, shows potent anti-tumor activity in a range of different cancer cell lines and now serves as a prototype for development of new retinoid related molecules (RRMs). The purpose of this study was to examine the effect and cellular targets of CD437 in the human metastatic melanoma cell lines FEMX-1 and WM239. We showed that treatment with CD437 led to cell cycle arrest and induced apoptosis through both the extrinsic- and intrinsic pathways (caspase 8, -9 and PARP cleavage) in both cell lines. Interestingly, apoptosis was induced independently of DNA-fragmentation in FEMX-1 cells, and appeared partially caspase-independent in the WM239 cells. Additionally, up-regulation of CHOP mRNA and cathepsin D protein expression, following retinoid treatment, suggests involvement of the endoplasmatic reticulum (ER) and lysosomes, respectively. Combination of suboptimal concentrations of CD437 and lexatumumab, a TRAIL death receptor-2 agonist, resulted in synergistic reduction of viable cells, along with increased PARP cleavage. These results indicate that CD437 has a strong anti-neoplastic effect alone and in combination with lexatumumab in melanoma cell lines.