MCT-1 expression and PTEN deficiency synergistically promote neoplastic multinucleation through the Src/p190B signaling activation

Multinucleation is associated with malignant neoplasms; however, the molecular mechanism underlying the nuclear abnormality remains unclear. Loss or mutation of PTEN promotes the development of malignant tumors. We now demonstrate that increased expression of the oncogene MCT-1 (multiple copies in T-cell malignancy 1) antagonizes PTEN gene presentation, PTEN protein stability and PTEN functional activity, thereby further promoting phosphoinositide 3 kinase/AKT signaling, survival rate and malignancies of the PTEN-deficient cells. In the PTEN-null cancer cells, MCT-1 interacts with p190B and Src in vivo, supporting that they are in proximity of the signaling complexes. MCT-1 overexpression and PTEN loss synergistically augments the Src/p190B signaling function that leads to inhibition of RhoA activity. Under such a condition, the incidence of mitotic catastrophes including spindle multipolarity and cytokinesis failure is enhanced, driving an Src/p190B/RhoA-dependent neoplastic multinucleation. Targeting MCT-1 by the short hairpin RNA markedly represses the Src/p190B function, improves nuclear structures and suppresses xenograft tumorigenicity of the PTEN-null breast cancer cells. Consistent with the oncogenic effects in vitro, clinical evidence has confirmed that MCT-1 gene stimulation is correlated with p190B gene promotion and PTEN gene suppression in human breast cancer. Accordingly, MCT-1 gene induction is recognized as a potential biomarker of breast tumor development. Abrogating MCT-1 function may be a promising stratagem for management of breast cancer involving Src hyperactivation and/or PTEN dysfunction.

MCF-10A cells were starved for 12 h and incubated with or without MG132 (50 M) for another 12 h before serum activation for 30 min. The effects of proteasome inhibition on AKT (ser473), PTEN and p53 were evaluated.

Immunoblot analysis
The active phosphorylation of AKT, PTEN, ERK1/2 and EGFR were studied in the MCF-10A cells activated for 30 min in DMEM/F12 complete medium after starving in DMEM/F12 basal medium for 24 h. The cellular response in late mitotic stage was analyzed by that MCF-10A cells were treated with nocodazole (50 ng/ml) for 18 h, washed with PBS and re-cultured in nocodazole-free medium for 1 h. The phosphorylation of Src, p190B and RhoA were examined when MCF-10A or MDA-MB-468 cells were activated with serum for 30 min after starving for 24 h. The protein samples were analyzed by SDS-PAGE and immunoblotting as previously described (31).

Immunofluorescence and fluorescence time-lapse microscopy
The cells were cultured in nocodazole-free media for 30 min after nocodazole treatment for 24 h.
Immunofluorescence study was conducted as described previously (28). The fluorescence images were captured using a Leica TCS NT confocal microscope (Leica) equipped with a 63x objective lens (HCX PLAPO lambda blue 63x NA 1.4 UV) and analyzed by Leica LAS AF software. For time-lapse microscopy, the cells (1x10 5 ) were cultured in a 0.17-mm ibidi -dish (ibidi) and incubated with or without Hochest 33342 dye (100 ng/ml) (Invitrogen). The images were recorded at 20-or 30-min intervals for 36 h using a Leica AF6000 LX fluorescence imaging system (Leica) equipped with a 20x objective lens (HCX PLAPO 20x NA 0.7) and analyzed by LASAF software.
The clarified lysates were reacted with pre-immunized IgG or p190B Ab for 4 h at 4 o C, incubated with 50 l protein A/G magnetic beads for 4 h at 4 o C and washed by RIPA buffer for 5 times before SDS-PAGE. The immune-reactive proteins were detected by HRP-conjugated EasyBlot anti-mouse or anti-rabbit secondary Ab (GeneTex).

Cytogenetic study
The MCF-10A cells were mitotic arrested with 0.1 g/ml colcemid (Calbiochem) for 4 h. Cells were harvested and incubated with pre-warmed 75 mM KCl at 37 •C for 40 min. Following hypotonic swelling, fresh fixative reagent (methanol:acetic acid = 3:1) was slowly dropped into cell pellets while gently tapping the tubes. Cell samples were fixed at room temperature for 10 min and re-fixed twice. The fixed samples were dropped onto slides and dry slides at 100•C for 25 min. The samples were stained with Wright's stain solution (MERCK) for 1 min. The chromosome number were analyzed for at least 100 metaphase cells.
ArrayCGH study and data analysis DNA samples (test and reference) were obtained from the control and MCT-1-overexpressing MCF-10A cell lines using genomic PUREGENE DNA purification kit (Gentra Systems). All samples were sheared by sonication and labeled with 532 nm-Cy3 and 635 nm-Cy5 dye-modified random primers. Labeled DNA samples of each group were combined and hybridized to a