Because the xenografted tumours responded differentially to DR despite similar decreases in insulin and IGF-1 levels, we determined whether the six cancer cell lines studied have differential requirements for these factors for their growth in tissue culture. Indeed, in cell lines that form DR-sensitive tumours (MDA-MB-231, MDA-MB-435, and SW620) insulin or IGF-1 caused a dose-dependent increase in cell numbers (). In contrast, cell lines that generate DR-resistant tumours (MCF10DCIS, U87-MG, PC3) grew in culture in an insulin and IGF-1-independent fashion ().
Constitutive PI3K activation correlates with tumour resistance to DR
These results suggested that the cancer cell lines forming DR-resistant tumours have a deregulation in an insulin/IGF-1activated signaling pathway, with the phosphatidylinositol 3-kinase (PI3K)/Akt pathway18
being an attractive candidate. Acting through receptor tyrosine kinases, insulin and IGF-1 recruit PI3K to the cell membrane19
, where its activity, which is antagonized by the PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumour suppressor, leads to the recruitment and activation of Akt, a serine/threonine kinase. In turn, Akt phosphorylates and regulates numerous targets that enhance cellular growth and inhibit apoptosis. Consistent with a deregulation in PI3K signaling in cell lines that form DR-resistant tumours, serum withdrawal for 1 or 24 hours did not affect Akt S473 phosphorylation, a marker of Akt activation, in MCF10DCIS, PC3, and U87-MG cells but did in MDA-MB-231, MDA-MB-435, and SW620 cells ().
Analysis of PTEN expression showed that PTEN loss could account for the serum-insensitive Akt activity20
in PC3 and U87-MG, but not, MCF10DCIS cells (). Activating mutations in the PIK3CA
gene, which encodes the catalytic subunit of PI3K, also lead to constitutive Akt activity in cancer cells21–24
. By sequencing in all the cell lines exons 9 and 20 of PI3KCA
that can be the sites of the “hot-spot” E545K and H1047R mutations21,25
, respectively, we found that the MCF10DCIS cells harbor a previously unknown H1047R mutation. None of the cell lines that form DR-sensitive tumors have lost PTEN function or carry PI3K activating mutations26
(). Furthermore, no other common oncogene-activating (e.g. RAS
) or tumour suppressor-inactivating (e.g. TP53
) mutation correlated with tumour cell sensitivity to insulin/IGF-1 in vitro or tumour sensitivity to DR in vivo26
(). Although PI3K can be directly activated by ras27
and this is necessary for the initiation of ras-driven tumours28
, the MDA-MB-231 and SW620 cell lines carrying activated RAS
alleles form DR-sensitive tumors (). These results imply that tumour resistance to DR correlates with constitutive activation (e.g. through PIK3CA
mutations or PTEN loss), rather than hyperactivation of the PI3K pathway.