Posttranslational regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in mouse PTEN null prostate cancer cells: Enhanced surface expression and differential O-glycosylation of MT1-MMP

Abstract

Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten). However, the role of PTEN inactivation in MT1-MMP expression in PC cells has not been examined. In this study, prostate epithelial cell lines derived from mice that are either heterozygous (PTEN(+/-)) or homozygous (PTEN(-/-)) for PTEN deletion or harboring a wild-type PTEN (PTEN(+/+)) were used to investigate the expression of MT1-MMP. We found that biallelic loss of PTEN is associated with posttranslational regulation of MT1-MMP protein in mouse PC cells. PTEN(-/-) PC cells display higher levels of MT1-MMP at the cell surface when compared to PTEN(+/+) and PTEN(+/-) cells and consequently exhibited enhanced migratory and collagen-invasive activities. MT1-MMP displayed by PTEN(-/-) cells is differentially O-glycosylated and exhibits a slow rate of turnover. MT1-MMP expression in PTEN(-/-) cells is under control of the PI3K/AKT signaling pathway, as determined using pharmacological inhibitors. Interestingly, rapamycin, an mTOR inhibitor, upregulates MT1-MMP expression in PTEN(+/+) cells via PI3K activity. Collectively, these data in a mouse prostate cell system uncover for the first time a novel and complex relationship between PTEN loss-mediated PI3K/AKT activation and posttranslational regulation of MT1-MMP, which may play a role in PC progression.

Fig 1

Differential expression of MT-MMP mRNA and MT1-MMP protein in mouse prostate epithelial cells with different PTEN status. (A) Total RNA from PTEN^+/+ (black bar) and PTEN^-/- (white bar) cells cultured in complete media were used for cDNA synthesis and subsequently real time PCR to quantify MT1-, MT2-, and MT3-MMP mRNA. Ct value of each gene was normalized by the GAPDH Ct value. Results shown are representative of two independent experiments. *, p <0.05; Student's t test. NS, not statistically significant. (B) Cell lysates from PTEN^+/+, PTEN^+/-, and PTEN^-/- cells were resolved by reducing 10% SDS-PAGE followed by immunoblotting analysis using mAb 3G4 against the catalytic domain of MT1-MMP (Mr ∼57-59 kDa). β-actin was used as a loading control. Asterisk shows non-specific bands.

Fig. 2

Elevated surface expression of MT1-MMP in PTEN^-/- cells. (A) PTEN^+/+ (lanes 1 and 2), PTEN^+/- (lane 3 and 4), and PTEN^-/- cells (lane 5 and 6) were seeded on 6-well plates and incubated with (+) or without (-) cell-impermeable biotin for 30 min on ice. Cell lysates from each cell were then incubated with avidin-conjugated beads and biotinylated proteins were resolved by reducing 10% SDS-PAGE followed by immunoblot analysis using mAb 3G4. The same blot was reprobed with an antibody against transferrin receptor (TFR), as a control. (B) Lysates from PTEN^+/+ and PTEN^-/- cells were resolved by reducing 7.5% SDS-PAGE followed immunoblotting analysis using mAb 3G4. (C) Lysates from PTEN^+/+ and PTEN^-/- cells were immunoprecipitated with pAb28209 to the cytosolic tail of MT1-MMP or non-immune rabbit IgG and protein-A conjugated agarose beads. The immunoprecipitates were resolved by reducing 7.5% SDS-PAGE followed immunoblotting analysis using mAb 3G4. Arrowheads on the right in panes B and C indicate the different species of MT1-MMP detected. Asterisk shows non-specific bands.

Fig. 3

Enhanced expression of MT1-MMP in lysates of wild type and PTEN^-/- mouse prostate tissues. Mouse PTEN^-/- prostate tumor (T₁ and T₂, lanes 4 and 6) and normal (N₁ and N₂, lanes 3 and 5) prostate lysates from different mice and lysates from cultured PTEN^+/+ (lane 1) and PTEN^-/- (lane 2) cells were resolved by reducing 8% SDS-PAGE followed by immunoblot analyses using mAb 3G4 to the catalytic domain. The antigen was detected using the TrueBlot™ ULTRA antibody against mouse IgG followed by chemiluminescence. Asterisks show non-specific bands.

Fig. 4

MT1-MMP is differentially O-glycosylated in PTEN^+/+, PTEN^+/- and PTEN^-/- cells. PTEN^+/+ (lanes 1 and 4), PTEN^+/- (lanes 2 and 5), and PTEN^-/- cells (lanes 3 and 6) were incubated with (+) or without (-) the O-glycosylation inhibitor BGN (2 mM) for 48 h. The cells were then lysed, and the lysates were resolved by reducing 7.5% SDS-PAGE followed immunoblot analysis using the mAb 3G4. Arrowheads on the right indicate the different species of MT1-MMP detected.

Fig. 5

Reduced turnover of MT1-MMP in PTEN^-/- cells. (A-D) PTEN^+/+ and PTEN^-/- cells were incubated with 50 μg/ml of cycloheximide (CHX) for 8 h. At each time point (0, 2, 4, and 8 h), the cells were lysed and the lysates were resolved by reducing 10% SDS-PAGE followed by immunoblotting analysis using mAb 3G4. A and B show blots derived from two independent experiments. C and D show the results of the densitometry analyses, as described in Materials and Methods. Values represent the relative amount of MT1-MMP at each time point relative to time zero (100%).

Fig. 6

Pro-MMP-2 activation, migration and invasiveness of PTEN^-/- cells. (A) Cultures of PTEN^+/+, PTEN^-/-, and HT1080 cells in 6-well plates were untreated (-) or treated (+) with ConA and the lysates were subjected to gelatin zymography. P, pro-MMP-2 (∼72 kDa), I, intermediate form of MMP-2 (∼66 kDa), A, active MMP-2 (∼62 kDa). Mo, mouse, Hu, human. (B) Confluent cultures of PTEN^+/+ and PTEN^-/- cells in 6-well plates were scratched, and incubated in complete media supplemented with 5 μg/ml mitomycin C for 8 h. The cell monolayers were then photographed. (C) PTEN^+/+ and PTEN^-/- cells were seeded on top of a type I collagen gel in the presence or absence of BB-94. After 14 days, 5 μm-thick frozen sections were stained with toluidine blue to identify the cells, indicated as black arrows. The experiments presented in panels A, B, and C were repeated at least three times with similar results.

Fig. 7

Effect of pharmacological inhibitors to PI3K, AKT, MAPK and mTOR on MT1-MMP expression. Serum starved PTEN^+/+ and PTEN^-/- cells were incubated (18 h) with (+) or without (-) the indicated doses of either PD 98,059 (A), LY 294,002 (B), Akt inhibitor IV (C) or rapamycin (D) in serum-free media. (E) Serum starved PTEN^-/- cells were untreated (-) or treated (+) with either LY 294,002 or PD 98,059 for 18 h. Then, the cultures received rapamycin (+) or vehicle (-) and were incubated for 18 h incubation. At the end of the incubation period, the cells were lysed, and the lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot analyses with the appropriate antibodies. The data presented in panels A-E were repeated at least three times with similar results.