The mannose 6-phosphate-binding sites of M6P/IGF2R determine its capacity to suppress matrix invasion by squamous cell carcinoma cells

Abstract

The M6P (mannose 6-phosphate)/IGF2R (insulin-like growth factor II receptor) interacts with a variety of factors that impinge on tumour invasion and metastasis. It has been shown that expression of wild-type M6P/IGF2R reduces the tumorigenic and invasive properties of receptor-deficient SCC-VII squamous cell carcinoma cells. We have now used mutant forms of M6P/IGF2R to assess the relevance of the different ligand-binding sites of the receptor for its biological activities in this cellular system. The results of the present study demonstrate that M6P/IGF2R does not require a functional binding site for insulin-like growth factor II for inhibition of anchorage-independent growth and matrix invasion by SCC-VII cells. In contrast, the simultaneous mutation of both M6P-binding sites is sufficient to impair all cellular functions of the receptor tested. These findings highlight that the interaction between M6P/IGF2R and M6P-modified ligands is not only important for intracellular accumulation of lysosomal enzymes and formation of dense lysosomes, but is also crucial for the ability of the receptor to suppress SCC-VII growth and invasion. The present study also shows that some of the biological activities of M6P/IGF2R in SCC-VII cells strongly depend on a functional M6P-binding site within domain 3, thus providing further evidence for the non-redundant cellular functions of the individual carbohydrate-binding domains of the receptor.

Figure 1. Ligand-binding properties of mutant forms of M6P/IGF2R

Left-hand panel, membrane extracts of SCC-VII cells expressing either wt or mutant M6P/IGF2R were incubated with either biotinylated IGF-II or BSA prior to the addition of avidin–Sepharose beads. The bound material was then subjected to immunoblotting analysis with anti-M6P/IGF2R antibodies. Right-hand panel, membrane extracts of SCC-VII cells expressing either wt or mutant receptor variants were incubated with phosphomannan–Sepharose beads. After washing with glucose 6-phosphate (G6P), the specifically bound proteins were eluted with M6P prior to analysis by immunoblotting with anti-M6P/IGF2R antibodies. T, total material applied to the beads. *A C-terminally truncated form of M6P/IGF2R [25].

Figure 2. Lysosomal enzyme delivery via the secretion-recapture pathway in SCC-VII cells expressing mutant M6P/IGF2R

SCC-VII cells expressing either wt or mutant M6P/IGF2R were cultured for 24 h in the presence or absence of 5 mM M6P. Parental and mock-transfected SCC-VII cells were analysed in parallel. Cell homogenates and conditioned medium were then analysed for their HEX activity. Results are means±S.E.M. for three to four independent experiments. *P<0.05, comparison of treated and untreated cells.

Figure 3. Invasive properties of SCC-VII cells expressing mutant forms of M6P/IGF2R

Matrigel invasion assays of SCC-VII cells expressing either wt or mutant M6P/IGF2R were performed using conditioned medium of fibroblasts as a chemoattractant. Mock-transfected cells were analysed in parallel. Parental SCC-VII cells were used as controls (set to 100%). Results are means±S.E.M. for at least three independent experiments. *P<0.05, compared with the mock-transfected cells.

Figure 4. In vitro growth of SCC-VII cells transfected with mutant M6P/IGF2R cDNAs

SCC-VII cells expressing either wt or mutant M6P/IGF2R were grown for 72 h under standard culture conditions prior to harvesting and cell number determination. Mock-transfected cells were analysed in parallel. Parental SCC-VII cells were used as controls (set to 100%). Results are means±S.E.M. for at least three independent experiments. *P<0.05, compared with the mock-transfected cells.

Figure 5. Anchorage-independent growth of SCC-VII cells transfected with mutant M6P/IGF2R cDNAs

SCC-VII cells expressing either wt or mutant M6P/IGF2R were cultured for 3 weeks in semi-solid medium before the median diameter of the colonies formed was determined. Mock-transfected cells were analysed in parallel. Parental SCC-VII cells were used as controls (set to 100%). Results are means±S.E.M. for at least three independent experiments. *P<0.05, compared with the mock-transfected cells.