Mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R): A putative tumor suppressor

Studies with gene knockout mice have demonstrated that M6P/IGF2R is an inhibitor of IGF2-dependent embryonic growth. Tumor-associated M6P/IGF2R mutations that may disrupt IGF2 binding have been described suggesting that the IGF2 antagonist action of M6P/IGF2R may also play a role in suppressing tumorigenesis; M6P/IGF2R has therefore been described as a putative tumor suppressor gene. Our studies focus on providing functional evidence to determine whether M6P/IGF2R can be considered a tumor suppressor in IGF2-dependent malignancies. To investigate the role of M6P/IGF2R as an IGF2 antagonist in breast cancer, M6P/IGF2R was overexpressed in IGF2-sensitive MCF7 cells. Recombinant IGF1- and IGF2-dependent proliferation was compared between control transfected cells and cells constitutively overexpressing wildtype or binding defective mutant M6P/IGF2R. Despite 5- to 10-fold overexpression of wildtype M6P/IGF2R, IGF2-dependent growth in either monolayer or in suspension culture was unaffected. To confirm this unanticipated negative result, MCF7 cell clones were engineered to overexpress wildtype M6P/IGF2R under the control of a doxycycline inducible promoter (iwt1 cells). Despite 10- to 15-fold induction of M6P/IGF2R expression with doxycycline, growth in the presence of IGF2 was unaltered when compared with control cells growing without doxycycline. Furthermore, the kinetics of IGF2-induced insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation was not modulated by doxycycline-induced M6P/IGF2R overexpression. Confocal microscopy confirmed that overexpressed M6P/IGF2R was located in the plasma membrane the appropriate intracellular compartments (Golgi and endosomes). We had previously demonstrated that affinity of IGF2 for M6P/IGF2R suppresses autocrine IGF2 activity. These data led us to hypothesize that M6P/IGF2R may operate to inhibit autocrine IGF2 within the secretory pathway but not efficiently suppress the action of IGF2 after it has been released from the cell. We transfected iwt1 cells to constitutively express IGF2 which allowed us to investigate the effect of M6P/IGF2R overexpression on growth rate, protection against apoptosis and IRS-1 phosphorylation status due to autocrine IGF2 signaling. We were unable to detect any effect of M6P/IGF2R overexpression on apoptosis or IRS-1 phosphorylation signaling but could demonstrate antagonism of IGF2-dependent growth in monolayer culture. Although the functional evidence at this time is insufficient to make the case that M6P/IGF2R is a tumor suppressor, the genetic evidence is compelling and justifies further experiments in various model systems.