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. 2009 Dec 1;69(23):8868-76.
doi: 10.1158/0008-5472.CAN-09-1968. Epub 2009 Nov 10.

Differential enhancement of breast cancer cell motility and metastasis by helical and kinase domain mutations of class IA phosphoinositide 3-kinase

Huan Pang  1 , Rory Flinn, Antonia Patsialou, Jeffrey Wyckoff, Evanthia T Roussos, Haiyan Wu, Maria Pozzuto, Sumanta Goswami, John S Condeelis, Anne R Bresnick, Jeffrey E Segall, Jonathan M Backer
Affiliations

"VSports" Differential enhancement of breast cancer cell motility and metastasis by helical and kinase domain mutations of class IA phosphoinositide 3-kinase

Huan Pang et al. Cancer Res. .

Abstract

Class IA (p85/p110) phosphoinositide 3-kinases play a major role in regulating cell growth, survival, and motility. Activating mutations in the p110alpha isoform of the class IA catalytic subunit (PIK3CA) are commonly found in human cancers. These mutations lead to increased proliferation and transformation in cultured cells, but their effects on cell motility and tumor metastasis have not been evaluated. We used lentiviral-mediated gene transfer and knockdown to produce stable MDA-MB-231 cells in which the endogenous human p110alpha is replaced with either wild-type bovine p110alpha or the two most common activating p110alpha mutants, the helical domain mutant E545K and the kinase domain mutant H1047R. The phosphoinositide 3-kinase/Akt pathway was hyperactivated in cells expressing physiologic levels of helical or kinase domain mutants. Cells expressing either mutant showed increased motility in vitro, but only cells expressing the helical domain mutant showed increased directionality in a chemotaxis assay VSports手机版. In severe combined immunodeficient mice, xenograft tumors expressing either mutant showed increased rates of tumor growth compared with tumors expressing wild-type p110alpha. However, tumors expressing the p110alpha helical domain mutant showed a marked increase in both tumor cell intravasation into the blood and tumor cell extravasation into the lung after tail vein injection compared with tumors expressing wild-type p110alpha or the kinase domain mutant. Our observations suggest that, when compared with kinase domain mutations in a genetically identical background, expression of helical domain mutants of p110alpha produce a more severe metastatic phenotype. .

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Figures

Figure 1
Figure 1. p110α expression, Akt phosphorylation and PIP3 production in cell lines expressing mutant bovine p110α in place of wild type
A. Anti-p110α immunoprecipitates were prepared from control MDA-MB-231 cells, cells infected with a lentiviral shRNA construct targeting human p110α, or cells in which human p110α was rescued with wild type bovine p110α. The immunoprecipitates were immunoblotted with anti-p110α (top panel) or anti-myc (lower panel) antibody. B. Control cells or stable MDA-MB-231 cells in which human p110α was replaced with wild type or mutant bovine p110α (helical domain: E545K; kinase domain: H1047R; kinase dead: KD) were immunoprecipitated with anti-p110α,β or δ antibodies. Immunoprecipitates or whole cell lysates were blotted for p110α or δ or p85 as indicated. C. Control cells or stable MDA-MB-231 cells in which human p110α was replaced with wild type or mutant bovine p110α were immunoprecipitated with anti-myc antibody, and blotted with anti-p110α (top panel) or anti-p85α (middle panel) antibodies. Whole cell lysates from these cell lines were blotted with anti β-actin (lower panel) as a loading control. D. Control MDA-MB-231 cells or cells in which human p110α was replaced with bovine wild type or mutant p110α were starved in starvation medium for 4h, and stimulated without or with 5nm EGF for 3min. Equal amount of cell lysate was separated by 10% SDS-PAGE and blotted for P-S473-Akt or total Akt. E. Control MDA-MB-231 cells or cells in which human p110α was replaced with bovine wild type or mutant p110α were serum starved overnight, fixed and stained with anti-PIP3 antibody as described. Phase contrast and immunofluorescence images for each cell line are shown.
Figure 2
Figure 2. Expression of p110α mutants increases protrusion and migration
A: Stable MDA-MB-231 cells expressing wild type or mutant (helical domain: E545K; kinase domain: H1047R) bovine p110α were seeded in 35mm dish coated with matrigel. After 12h to allow adhesion, the cells were starved for 4h, and stimulated with 2.5nm EGF. Time-lapse images were collected every 20s. The surface area of each cell was measured using ImageJ, and normalized to the initial cell area. The data are the mean ± SEM from 15–20 cells. B. Cells were plated collagen-coated transwell chambers, and incubated for 4h without or with EGF in the lower chamber. The cells were fixed and stained with DAPI, and the number of cells on the lower filter surface was counted. The data are the mean ± SEM from 3 experiments. C: Monolayer cultures of stable MDA-MB-231 cells expressing wild type or mutant bovine p110α were wounded with a P200 pipette tip. Phase-contrast images of the wound area were taken at the 0h and 20h. Images from the 0h and 20h incubations are shown. D: The data are the Mean ± SEM, and statistical significance were determined using a two-tailed students t-test. *: p<0.05; **: p<0.01 compared with cell expressing wild type p110α.
Figure 3
Figure 3. Expression of the helical domain mutant increases chemotaxis
Stable cells were placed in Dunn chambers in the presence or absence of EGF. Cells migration was recorded by time-lapse video microscopy, and analyses of migration and chemotaxis was performed using Mathematica notebooks written and provided by Professors GA Dunn and GE Jones, King’s College London (18). The bar height represents the proportion of cells moving in a particular direction, and the arrow and the shading indicate the mean direction of cell migration and its 95% confidence interval, respectively. A. Chemotaxis of control MDA-MB-231 cells in the absence or presence of a 0 to 5nm EGF gradient. B. Chemotaxis of cells expressing wild type or mutant bovine p110α in a 0–5nm EGF gradient. 40–50 cells were counted for each group. p < 0.005 helical domain mutant (E545K) vs. wild type; p < 0.05 helical domain mutant (E545K) vs. kinase domain mutant (H1047R) in the Moore test. C. Chemotaxis of helical domain and kinase domain mutants in a 0 to 0.5nm EGF gradient. p < .001 in the Moore test.
Figure 4
Figure 4. The helical domain mutation enhances tumor growth and in vivo intravasation
Cell lines expressing wild type or mutant p110α were injected into the right mammary fat pads of SCID mice. A. Spontaneous tumor size was recorded every week. **: p <0.01 helical domain (E545K) vs. kinase domain (H1047R). B. Blood burden (intravasation) experiments were performed once the tumor volumes reached 1.2–1.3cm. Adherent tumor cells were counted 14 days after plating of the blood samples. Data are normalized for the blood volume for each sample. n=6–11 mice per mutant type. **: p<0.01 E545K vs. wild type; *: p<0.05 H1047R vs. wild type; ***: p<0.01 E545K vs. H1047R in Mann-Whitney U test.
Figure 5
Figure 5. Tail vein injection of cells expressing mutant p110α lead to increased lung metastases
Stable cells expressing wild type or mutant p110α (helical domain: E545K; kinase domain: H1047R) were injected into the lateral tail vein of SCID mice. Mice were sacrificed 7 weeks after tail vein injection. A: Lungs from control and mutant p110α mice. B: Lung metastases were visualized by H&E staining. C: The area of lung metastases were determined by Image J. ***, p<0.001, relative to wild type.
Figure 6
Figure 6. Increased lung metastasis in cells expressing helical domain versus kinase domain mutants of p110α
Stable cell lines were injected into the lateral tail vein of SCID mice as in Figure 5. Mice were sacrificed 4 weeks after tail vein injection. A: Lung metastases were visualized by H&E staining. B: The area of lung metastases were determined by Image J. C: The number of metastatic foci were determined by Image J. ***: p<0.001, relative to E545K.
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References

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