Abstract
To analyze mechanisms for cancer metastasis, we established high metastatic sublines from mouse Lewis lung cancer (P29) by repeated injection. Sublines established from the two subclones H7 and C4 commonly exhibited increased proliferation and invasion activity and reduced expression of ganglioside GM1, although they showed different preferences in their target organs of metastasis. The high metastatic sublines secreted higher levels of activated matrix metalloprotease (MMP)-9 as well as pro-MMP-9 in the culture medium than the parent lines. Furthermore, they contained MMP-9 at the glycolipid-enriched microdomain (GEM)/rafts fractionated by the sucrose density gradient ultracentrifugation of Triton X-100 extracts, whereas the parent cells showed faint bands at the fraction. When high metastatic sublines were treated with methyl-beta-cyclodextrin, their invasion activities were dramatically suppressed, and the MMP-9 secretion was also suppressed. All these results indicated that GEM/rafts play crucial roles in the increased invasion and high metastatic potential. To clarify the implication of reduced GM1 expression, low GM1-expressing cell lines were established using an RNA interference-expression vector of the GM1 synthase. Low GM1-expressing cell lines showed increased proliferation and invasion, enrichment in the GEM/rafts, and increased secretion of MMP-9. Among adhesion molecules, only integrin beta1 was detected in GEM/rafts with stronger intensity in high metastatic lines and low GM1-expressing cells. Taken together, integrins seemed to be enriched in the GEM/rafts by reduced GM1 levels, and subsequently MMP-9 was recruited to the GEM/rafts, resulting in its efficient secretion and activation, and eventually in the increased invasion and metastatic potentials.
Highlights
Roles of proteolysis and proteases in cancer metastasis have been dramatically elucidated [6]
matrix metalloproteases (MMP)-9 bands (105 kDa) appeared mainly in the raft fraction of high metastatic lines, but only faint or no bands were present in that of the parent lines. These results suggested that localization of MMP-9 to the glycolipid-enriched microdomain (GEM)/rafts fraction resulted in the increased MMP-9 secretion and activation, leading to the increased invasion activity
Because ganglioside GM1 has been considered to be one of the constituents of lipid rafts or microdomains, where cellular signals are exchanged on the cell membrane [12], these results strongly suggest that GM1 was involved in the regulation of MMP-9 secretion and activity
Summary
Cell Lines and Culture—P29, a low metastatic subline (with subcutaneous inoculation) of the Lewis lung cancer cell line, was subcloned to establish monoclonal sublines H7 and C4. After washing three times with PBST, the blots were incubated for 1 h with goat anti-rabbit IgGs or goat anti-mouse IgGs conjugated with horseradish peroxidase (Cell Signaling) (1:4000). Cells were solubilized in the Triton X-100-containing lysis buffer (Cell Signaling) at room temperature, and the lysates were incubated with 10 l of anti-integrin 1 (2 g of IgG) or normal rabbit IgG for 2 h at room temperature. After incubation in PBS with 5% bovine serum albumin, the membranes were probed with CTB/biotin (1:200), and bands were detected with an ABC kit (Vector Laboratories) and immunostained with horseradish peroxidase-1000 kit from Konica (Tokyo, Japan). Statistical Analysis—Statistical significance of data were determined using Student’s t test
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