Phosphoinositide 3-hydroxide kinase blockade enhances apoptosis in the Ewing's sarcoma family of tumors

Ewing's sarcoma family of tumors (ESFTs) affects patients between the ages of 3 and 40 S ears, with most cases occurring in the second decade of life. These tumors contain a characteristic translocation, t(11;22), that produc es a unique fusion protein, EWS/FLI-1. EWS/FLI-1 transforms mouse fibroblas ts; this transformation requires intact EWS and FLI-1 domains as well as th e insulin-like growth factor-I receptor (IGF-IR). The IGF-IR is a well-dcsc ribed transmembrane tyrosine kinase receptor that modulates transformation, cell growth, and survival. IGF-IR survival signaling is mediated through t he downstream activation of phosphoinositide 3-OH kinase (PI 3-K) and Akt. Apoptosis, programmed cell death, progresses from a central death signal to a caspase cascade, including activation of caspase-3, Because the IGF-IR h as been shown to play a role in the transformation and growth of ESFTs, we wanted to determine the role of downstream molecules in the cellular respon se to doxorubicin treatment. Doxorubicin increased caspase-3 activity in tw o ESFT cell lines, TC-32 and TC-71. Concomitant treatment of the doxorubici n-treated cells with IGF-I reduced caspase-3 activity 8-fold in TC-32 and 4 -fold in TC-71, To determine whether PI 3-K has a role in IGF-I-mediated su rvival in ESFTs, PI 3-K; was then inhibited with wortmannin and LY294002. D oxorubicin treatment reduced cell number and enhanced apoptosis in PI 3-K i nhibited cells compared with noninhibited cells. Akt, a serine/threonine ki nase activated downstream of PI 3-1;, was investigated to determine whether its constitutive activation would render ESFT cells more resistant to doxo rubicin. A constitutively activated Akt was stably transfected into ESFT an d those cells with high levels of expression demonstrated increased resista nce to doxorubicin-induced caspase-3 activation. These results indicate tha t ESFT cell lines use an IGF-LR initiated signaling pathway through PI 3-K; and Akt for survival when treated with doxorubicin.