The role of JAK2, STAT3 and ERBB2 in ovarian cancer
Creators: name: Studd, James
Publisher: name: Imperial College London
Year: 2013

description: Background Ovarian cancer is the most lethal gynaecological malignancy, accounting for an estimated 140,000 deaths per year worldwide. Five year survival rates have not increased significantly in the last 10 years and the acquisition of resistance to chemotherapy remains a significant barrier to improving patient survival. Isogenic cell line models of in vivo acquired resistance to chemotherapy were used to examine cellular responses to cisplatin and identify differences between sensitive and resistant pairs that might be exploited to sensitise cells to treatment. Results Microarray analysis of the isogenic paired sensitive/resistant high grade serous ovarian cell lines PEO1 and PEO4 revealed IL6 expression is induced by cisplatin exposure. This result was replicated by QRT-PCR and validated in the additional isogenic pair PEA1/PEA2. Western blotting demonstrated the lack of a correlation between IL6 expression and phosphorylation of either Y1007/1008 JAK2 or Y705 STAT3 levels, suggesting IL6 is not driving the constitutive activation of these proteins. Cells did however display dose dependant changes in STAT3, JAK2 and ERBB2 activation in response to cisplatin that differed between sensitive and resistant isogenic pairs. Both sensitive clones increased their activation of JAK2 and ERBB2 when exposed to low dose, 2µM, cisplatin but reversed these increases at higher concentrations. Resistant clones, PEO4 and PEA2, experienced no low dose increases in ERBB2 JAK2 or STAT3 activation instead reducing the activation of these proteins with greater sensitivity to cisplatin dose. Common to all cell lines was a high degree of correlation in the levels of activated JAK2 and ERBB2. Interfering with cisplatin dependent STAT3 deactivation using IL6 treatment was able to both sensitise cells and reduce cisplatin IC50, suggesting a functional role for STAT3 in both response, and acquired resistance, to cisplatin. Overexpression and knockdown of STAT3 demonstrated it promotes proliferation and the expression of cyclin D1 and BCL xL/S. STAT3 knockdown increased cisplatin resistance as quantified by IC50 whereas STAT3 overexpression was able to potentiate cisplatin induced apoptosis and decrease cisplatin IC50. Similarly the overexpression and knockdown of JAK2 demonstrated it also promotes proliferation, in part by regulating the activity of STAT3. The inhibition of JAK2 activity also increased resistance to cisplatin; small molecule inhibition of JAK2 both lowered background levels of apoptosis as well as attenuating cisplatin induced apoptosis. In common with STAT3 ablation JAK2 knockdown also increased cisplatin IC50. Surprisingly knockdown, overexpression and inhibition of JAK2 were all associated with changes in the activation of ERBB2. Knockdown and inhibition were associated with decreases in Y1248 phosphorylated ERBB2 whereas overexpression was associated with an increase, changes in activation appear to be driven by changes at the level of total protein. GP130 was investigated for due to its role in IL6 signalling and STAT3 activation, mRNA overexpressed was detected in the resistant pair of 2/3 isogenic cell lines. GP130 overexpression was associated with growth promotion and cisplatin resistance as revealed by siRNA knockdowns, which had no effect in cisplatin sensitive non overexpressing cells lines. Knockdown also revealed different pathways to constitutive STAT3 activation, in each high grade serous line assessed there was no effect on pSTAT3 levels, which were almost completely abolished in SKOV3. RNAi of GP130 was also associated with an increase in ERK1/2 activation which was recapitulated by JAK2 knockdown, inhibition as well as cisplatin and doxorubicin exposure. Treatment with a MEK1/2 inhibitor was able to reverse cisplatin and doxorubicin dependent activation and attenuate cytotoxic induced apoptosis. MEK1/2 inhibition was associated with an increase in JAK2, pSTAT3 and pERBB2 which was capable of reversing cisplatin dependent down regulation of these protein, highlighting a mutual feedback mechanism between the GP130/JAK2 and MAPK pathways. Central Conclusion Transcriptional regulation of JAK2 in response to cisplatin exposure drives differential behaviour of paired isogenic cell lines. Greater sensitivity of cisplatin resistant cells lines, in their deactivation of STAT3 and ERBB2 is regulated by their greater extent of JAK2 downregulation upon cisplatin exposure. Downregulation of JAK2 and its commensurate reduction in STAT3 activation were associated with reduced proliferation rates, rendering cells in a state refractory to cisplatin toxicity. This may be due to either or both of reducing the accumulation of DNA double stranded breaks associated with cell division and allowing more time for the repair of single stranded DNA adducts before cell division. While STAT3 has been suggested as a target for adjuvant chemotherapy, data presented here suggests that in combination with cisplatin STAT3 abrogation might actually reduce the effectiveness of treatment.