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Angiogenesis is a crucial factor in tumour growth and invasion, and the formation of metastases. Clinical studies have suggested a specific role of angiogenesis and its effectors in ovarian cancer development (Abu-Jawdeh et al., 1996).

In order to assess the effect of MEK5 and ERK5 inhibitor (BIX02189 and XMD8-92 respectively) treatment upon the angiogenic potential of A2780- WT, A2780-ADR and A2780-CP70 ovarian cancer cell lines, a co-culture assay of NHDF/HDMEC/A2780s was performed. After plating the fibroblast cells on day 1, the ovarian cancer cells were added onto the fibroblast monolayer on day 3. HDMECs were added to the NHDF/ovarian cancer cells after 24 hours. On day 5, the co-culture of NHDF/HDMECs/A2780s was treated with BIX02189 or XMD8-92 and incubated at 37 ºC until day 8. Treatment of the co-culture with inhibitors was repeated and incubated in 37 ºC for 48 hours. On day 10, the co-culture was fixed. Furthermore, a NHDF/HDMEC co-culture assay without tumour cells was run as a control.

It was found that tumour cells induced tube-formation in the NDHF/HDMEC/A2780 co-culture assay compared to the co-culture without cancer cells (Figure 5.11). Moreover, the inclusion of doxorubicin and cisplatin resistant cells in the co-culture assay prompted the development of more networked capillary vessels compared to the basal control assay and the A2780-WT sensitive tumour cell assay, which could mean that the expression levels of angiogenic factors (VEGF-A, B, C and D) from tumour cells are reflective of the degree of aggressiveness of the tumour cells (Figure 12d).

Quantification of total tube length revealed that the MEK5 inhibitor BIX02189 reduced tubule formation in the doxorubicin and cisplatin resistant cell lines by 3-fold respectively compared to non-treated cells (Figure 5.11b).

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In addition, treatment with the ERK5 inhibitor XMD8-92, decreased tube- formation more than 2-fold in A2780-ADR cells and A2780-CP70 cells compared to untreated cells. The decrease in the level of tube formation in resistant ovarian cancer cells after treatment with MEK5/ERK5 signalling pathway inhibitors, BIX02189 and XMD80-92, when compared to the sensitive ovarian cancer cells or basal control assay without tumour cells could indicate that ERK5 plays a role in resistant cancer cells and suggests that ERK5 is responsible in resistant cancer cell lines for inducing more capillary like-structures.

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Figure 5.11. The effect of MEK5/ERK5 inhibitors, BIX02189 and XMD8-92, on tumour angiogenesis in a HDMEC/NHDF/A2780s co-culture assay. (A) NHDF were seeded in

fibroblast growth medium at 20,000 cells per well on gelatine-coated 24-well plates and incubated for 48 hours. On day 3, A2780-WT, A2780-ADR and A2780-CP70 cells were plated at 5,000 cells per well on the NHDF layer and incubated for 24 hours. On day 4 of the assay, HDMECs were plated at 45,000 cells per well onto the confluent NHDF and A2780s layers with EBM MV2 basal medium FGM. On day 5, co-culture media were changed to EBM MV2 basal medium containing 1% (v/v) FCS and treated with indicated amount of BIX02189 and XMD8-92, as shown in the treatment schedule (C).The treatment was repeated on day 8. On day 10, cells were fixed and stained as described (section 2.2.9.3.3), and total tube length was quantified using AngioQuant image analysis software. (B) Data is presented as total tube length compared to basal. This result is typical of two independent experiments (**p<0.01).

B

C

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Figure 5.12 mRNA expression of in ovarian cancer cells. The bar chart shows RT-PCR

analysis of (A) mdr-1, (B) abcg2, (C) gst-pi and (D) vegf-a, vegf-b, vegf-c and vegf-d mRNA expression in A2780-WT, A2780-CP70 and A2780-ADR. The ovarian cancer cells RNA were extracted and cDNA prepared. Data were analysed by the ∆∆Ct value method and the expression was normalized to GAPDH expression and illustrated as fold change.

C

D

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5.6 Discussion

The purpose of conducting the experiments detailed in this chapter was to assess the role of the MEK5/ERK5 signalling pathway in doxorubicin and cisplatin resistant ovarian carcinoma cells by using the small molecule kinase inhibitors BIX02189 and XMD8-92.

In this study, the ovarian carcinoma cell lines (A2780-WT, A2780-ADR and A2780-CP70) were exposed to doxorubicin and cisplatin to assess the effect of these chemotherapeutic drugs on ERK5 activation.

Increased ERK5 activation was observed in the resistant cell lines in comparison with the sensitive cells which mean that the MEK5/ERK5 signalling pathway could play a role in drug resistance in ovarian carcinoma (Figure 5.2).

Furthermore, the resistant cells exposed to chemotherapeutic agents displayed increased ERK5 activation, which is considered an important contributor to the cell survival mechanism, suggesting that doxorubicin and cisplatin activate survival pathways in ovarian carcinoma cells (Wang and Tournier, 2006).

ERK5 activation is increased in response to growth factors (Mody et al., 2003). Thus, Figure 5.4a, b and c showed that phosphorylation of ERK5 increased in response to a variety of growth factors in all three ovarian cancer cell lines. Receptor tyrosine kinases of the ErbB family are activated when it interacts with ligands of the EGF family (Wang and Tournier, 2006).

Resistance to chemotherapy, metastasis and poor prognosis in cancer are associated with over expression of EGFR receptors (Wang and Tournier, 2006). EGFR is induced via epidermal growth factor (EGF) and other related ligands, whereas EGFR-2, 3 and 4 are activated by EGF-like ligands called neuregulins (NRGs) (Esparis-Ogando et al., 2002a). Analysis of ERK5

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activation in human derived cancer cell lines such as breast cancer has illustrated that constitutive activation of ERK5 is associated with activated forms of ErbB2, 3 and 4 (Esparis-Ogando et al., 2002a). Knockdown of ERK5 via siRNA induced apoptosis and decreased chemo resistance in HL- 60 acute myeloid leukaemia cells (Hayashi et al., 2005b).

In this chapter, EGF and NRG-1 were the most potent ligands that increased ERK5 activation in ovarian cancer cell lines (Figure 5.4). EGFR was activated in ovarian cancer cells in response to EGF. The gene expression of

erbb1 and erbb3 were increased in ovarian cancer resistant cell lines which

demonstrated their role in poor prognosis and resistance to chemotherapy agents in cancer patients (Figure 5.5c).

Inhibition of the MEK5/ERK5 signalling pathway by the small kinase inhibitors BIX02189 and XMD8-92 in combination with doxorubicin or cisplatin was investigated in doxorubicin and cisplatin resistant ovarian cancer cell lines (Figure 5.6 and 5.7). Combination therapies using chemotherapeutic drugs (doxorubicin and cisplatin) with MEK5/ERK5 signalling pathway inhibitors (BIX02189 and XMD8-92) or EGFR inhibitor (lapatinib) were applied to evaluate the effect of these combinations on the resistance of A2780 ovarian cancer cell lines.

BIX02189, XMD8-92 or lapatinib in combination with doxorubicin shifted the resistance in doxorubicin-resistant cells compared to cells treated with doxorubicin alone (Figure 5.9). The inhibition of EGFR combined with doxorubicin in A278-ADR cells restored sensitivity to doxorubicin to approximately the same level as the parental cell line (Figure 5.9). Moreover, treatment of cisplatin-resistant cells with a combination of lapatinib and cisplatin restored sensitivity to cisplatin to approximately the level of the parental cell line (Figure 5.10).

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Different mechanisms have been considered for doxorubicin resistance, including the expression of many membrane efflux pumps; the gene expression of MDR-1 and ABCG-2 in A2780-ADR cells was increased more than 3500-fold and 20-fold respectively in comparison with A2780-WT (Gottesman et al., 2002) (Figure 5.12a and b). Furthermore, increased drug metabolism enzymes such as glutathione S-transferase P1 (GSTP1) affect doxorubicin cytotoxicity in cancer and the gene expression of GSTP1 in A2780-ADR cells was increased compared to the parental cell line (Harbottle et al., 2001) (Figure 5.12c).

There are several mechanisms implicated in cisplatin resistance including increased DNA repair, decreased drug accumulation and increased detoxification via GSTP1 which is considered as a key limiting factor in cisplatin cytotoxicity (Pasello et al., 2008). Gene expression of GSTP1 in A2780-CP70 cells has been investigated and showed an increase compared to parental cell line (Figure 5.12c).

Thus in future works, inhibition of certain ABC transporters such as MDR-1 and ABCG-2 by MEK5/ERK5 could be a mechanism to assess the role of the MEK5/ERK5 signalling pathway in the regulation of chemoresistance in tumour cells that overexpress many ABC transporters. These findings suggested that the MEK5/ERK5 signalling pathway, along with EGFR, could be a possible therapeutic target to reduce drug resistance in ovarian cancer.

Deletion of the erk5 gene revealed its involvement in tumour associated angiogenesis and its effect in the vasculature development of melanoma and lung carcinoma xenograft (Hayashi et al., 2005b). In addition, deletion of

erk5 in flank region of mice reduced the number of large blood vessels in a

growing tumour (Hayashi et al., 2005b).

In this chapter, the role of the MEK5/ERK5 pathway in tumour angiogenesis was assessed by inhibition of this cascade via BIX02189 and XMD8-92

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inhibitors in a NHDF/HDMEC/A2780s co-culture in vitro angiogenesis assay.

Initially, embedding ovarian cancer cells in either sensitive or resistant state in NHDF/HDMEC co-culture revealed that tumour cells induced more capillary network vessels compared to co-cultures without tumour cells (Figure 5.11). Previous data from our group revealed that ERK5 is required for AKT phosphorylation, and also involved in VEGF-mediated survival and tubular morphogenesis in HDMECs (Roberts et al., 2010c). The doxorubicin or cisplatin resistant cells triggered more tubular formation compared with A2780-WT cells which demonstrates the aggressiveness of resistant cells.

The ability of tumour cells to increase tube-formation relates to increased secretion of VEGF-A to induce endothelial cells and increase tubule formation (Figure 5.12d). Treatment of the co-culture with MEK5 and ERK5 inhibitors (BIX02189 and XMD8-92) revealed that tube formation was reduced, and this effect was more evident in resistant cancer cells.

These data suggest that ERK5 plays a role in tumour angiogenesis and is required in resistant cancer cells to induce networked blood vessels; this may provide a possible means for therapeutic inhibition of angiogenesis in vivo. Future study should aim to investigate the effect of siRNA-mediated silencing of MEK5 and ERK5 expression in a NEHDF/HDMEC/cancer cells co-culture to further validate this model.

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6.1 The role of ERK5 signalling axis in tumour angiogenesis