Decreased expression of the mannose 6-phosphate/insulin-like growth factor-II receptor promotes growth of human breast cancer cells

Abstract

Background: Loss or mutation of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) has been found in breast cancer. However, whether or not decreased levels of functional M6P/IGF2R directly contribute to the process of carcinogenesis needs to be further verified by functional studies.

Methods: In this study, using viral and ribozyme strategies we reduced the expression of M6P/IGF2R in human breast cancer cells and then examined the effect on growth and apoptosis of these cells.

Results: Our results showed that infection of MCF-7 cells with the adenovirus carrying a ribozyme targeted against the M6P/IGF2R mRNA dramatically reduced the level of transcripts and the functional activity of M6P/IGF2R in these cells. Accordingly, cells treated with a ribozyme exhibited a higher growth rate and a lower apoptotic index than control cells (infected with a control vector). Furthermore, decreased expression of M6P/IGF2R enhanced IGF-II-induced proliferation and reduced cell susceptibility to TNF-induced apoptosis.

Conclusions: These results suggest that M6P/IGF2R functions as a growth suppressor and its loss or mutation may contribute to development and progression of cancer. This study also demonstrates that adenoviral delivery of the ribozyme provides a useful tool for investigating the role of M6P/IGF2R in regulation of cell growth.

Figure 1

Hammerhead ribozyme structure and target sequence of the M6P/IGF2R mRNA (A) and ribozyme activity against M6P/IGF2R RNA in vitro (B). ³²P-labeled RNA (40 nM) containing the M6P/IGF2R target sequence was incubated with the hammerhead ribozyme (60 nM) and the reaction products were separated on a polyacrylamide gel. The ribozyme cleaves the M6P/IGF2R target efficiently in vitro. Lane 1: Unmatched target alone; Lane 2: Unmatched target incubation with ribozyme at 12 hours; Lane 3: Specific target alone; Lane 4: Specific target incubation with ribozyme at time 0; Lane 5–7: Specific target incubation with ribozyme at 1, 2 and 12 hours.

Figure 2

RT-PCR detection of the M6P/IGF2R ribozyme expressed in MCF-7 cells. MCF-7 cells were infected with Ad.GFP.Rz-IGF2R or Ad.GFP. Seventy two hrs post infection, cells were collected and total RNA extracted. The presence of the ribozyme was detected by RT-PCR using primers specific to the ribozyme. Lane 1: Cells infected with Ad.GFP; Lane 2: cells infected with Ad.GFP.Rz-IGF2R.

Figure 3

Effect of the ribozyme on the level of M6P/IGF2R mRNA in MCF-7 cells. A: a representative RT-PCR result showing the levels of M6P/IGF2R (lanes 3 and 4) and the corresponding amounts of β-actin mRNA (lane 1 and 2) in ribozyme-treated (lane 1, lane 3) and in control cells (lane 2, lane 4). B: bar chart showing percent change in M6P/IGF2R mRNA of ribozyme-treated cells relative to the mRNA fraction in control cells (normalized to β-actin). Total RNA was isolated from cultured MCF-7 cells infected with Ad.GFP/Rz-IGF2R or Ad.GFP for 72 hrs, and then subjected to RT-PCR using M6P/IGF2R specific primers and β-actin as an internal standard. The data are the mean ± SE of three separate experiments (n = 3, ^*, p < 0.05 versus control).

Figure 4

Effect of the ribozyme on the internalization of ¹²⁵I-labeled IGF-II (A). Ribozyme-treated or control cells were incubated in medium containing ¹²⁵I-labeled IGF-II with or without excess unlabeled IGF-II. Following incubation, cells were washed and cell-associated radioactivity was determined using a γ counter. Determination of binding (B) and endocytosis (C) of M6P-containing proteins in MCF-7 cells. The M6P-bearing enzyme β-glucuronidase was used as a probe. The M6P-inhibitable binding was measured by incubating saponin-peameabilized cells with β-glucuronidase in the presence or absence of M6P. The M6P-inhibitable uptake was determined by incubating cells with medium containing β-glucuronidase in the presence or absence of M6P. Following incubation, cells were washed extensively, and cell-associated β-glucuronidase was measured fluorometrically. Each point, the mean of three separate experiments (n = 3; ^*, p < 0.05; versus control).

Figure 5

Effects of the ribozyme on the proliferation of MCF-7 cells. Cells were infected with Ad.GFP/IGF2R-Rz or Ad.GFP treated with or without IGF-II and then analyzed by MTT assay. Each point, the mean of three separate experiments (n = 3; ^*, p < 0.05; ^**, p < 0.01 versus control).

Figure 6

Effects of the IGF2R ribozyme on the apoptosis induced by TNF in MCF-7 cells. Cells were infected with Ad.GFP (control, left panels) or Ad.GFP/Rz-IGF2R (right panels). 72 hrs post infection, cells were treated with TNF for one day and then cell death was examined using a fluorescence microscope. Upper panels: Cells were stained with Hoechst dye for nuclei and observed under 480 nm blue-fluorescent light. The bright blue spots are the nuclei of apoptotic cells. Lower panels: Cells were stained with Propidium Iodide dye for nuclei and observed under 565 nm red-fluorescent light. The red spots are the nuclei of dead cells.

Figure 7

Effect of the ribozyme on the apoptosis induced by TNF in the MCF-7 cells. Cells were infected with Ad.GFP/IGF2R-Rz or Ad.GFP, treated with TNF and then cell viability was analyzed by MTT assay (A) and the apoptotic cells were assayed using the cell death ELISA assay (B). Each point, the mean of three separate experiments (n = 3; ^*, p < 0.05, versus control).