Phosphatidylinositol phosphate 5-kinase Iγi2 in association with Src controls anchorage-independent growth of tumor cells

Abstract

A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

Keywords: Anchorage-independent Growth; Cancer Biology; Cell Growth; Oncogene; Phosphatidylinositol; Src; Talin.

FIGURE 1.

PIPKIγ/PIPKIγi2 is required for anchorage-independent growth of tumor cells. A, schematic diagram of PIPKIγi2 showing the domain organization and the specific amino acid sequence at the C terminus. Red letters indicate Src phosphorylation sites crucial for talin binding. B–D, 48 hours after siRNA transfection, cells were cultured in soft agar for 2–4 weeks. Immunoblotting shows the knockdown of PIPKIγ and PIPKIγi2. Representative images of colonies developed by T47D cells are shown. E, for cell proliferation assay, MDA-MB-231 cells after siRNA transfection were seeded into 12-well culture plates (1,000 cells/well). Cells were counted manually every second day. F, 24–36 h after siRNA transfection, cells were retransfected with plasmids for expression of GFP-PLCδ-PH or GFP-PLCδ-PH mutant. Cells were processed for IF study after overnight culture (scale bar, 10 μm). G, siRNA was used to knock down endogenous PIPKIγi2 from MDA-MB-231 cells or MDA-MB-231 cells expressing siRNA-resistant wild type PIPKIγi2 (PIPKIγi2WT) or its kinase dead mutant (PIPKIγi2KD). Cells were cultured in soft agar 48 hours after transfection. Immunoblotting shows the knockdown of endogenous PIPKIγi2 in the cells. The expression of siRNA-resistant PIPKIγi2WT or PIPKIγi2KD was confirmed by immunoblotting using antibodies specific for PIPKIγi2 or HA tag. All the values are means ± S.D. from three-independent experiments. The error bars represent S.D. (p values are indicated).

FIGURE 2.

Overexpression of PIPKIγ/PIPKIγi2 promotes anchorage-independent growth. A, immunoblot showing the expression of HA-tagged PIPKIγ variants into MDA-MB-231 cells. B and C, oncogenic growth of MDA-MB-231 cells expressing different PIPKIγ variants were examined by counting the colonies developed after 2–3 weeks of culture in soft agar as described above. D, MDA-MB-231 cells expressing low and high levels of PIPKIγi2 were selected, and their oncogenic growth on soft agar was monitored as described above. E, siRNA was used to knock down ectopically expressed PIPKIγi2, and its effect on anchorage-independent growth was examined as described above. F, for cell proliferation assay, MDA-MB-231 overexpressing PIPKIγi1 or PIPKIγi2 was seeded into 12-well culture plates (1,000 cells/well). Cells were counted manually every second day. G, MCF-7 cells overexpressing PIPKIγi2 were cultured in soft agar for 21 days before counting the developed colonies (at least 10 randomly selected areas were used for counting the colonies). All the values are means ± S.D. from three-independent experiments. The error bars represent S.D. (p values are indicated).

FIGURE 3.

PIPKIγi2 regulates Src activation downstream of growth factor receptors and integrins. A and B, MDA-MB-231 and T47D cells after siRNA transfection for PIPKIγ or PIPKIγi2 knockdown were incubated in suspension condition, and the activation status of Src, FAK, and ERK1/2 was examined by immunoblotting using their specific antibodies. ConsiRNA, control siRNA. C, MDA-MB-231 cells expressing different PIPKIγ variants were incubated in suspension condition before harvesting the cells to examine the activation status of signaling molecules. D, siRNA was used to knock down the expression of ectopically expressed PIPKIγi2 before examining the activation level of Src and other molecules in suspension condition. E, mock or PIPKIγi2-overexpressing MDA-MB-231 cells were incubated in suspension condition (Susp.) before harvesting the cells at different time points to examine the activation level of Src. F, MDA-MB-231 cells after siRNA transfection for PIPKIγ or PIPKIγi2 knockdown were serum-starved overnight before stimulating with EGF (50 ng/ml) or 10% FBS. Cells were harvested at different time points to examine the activation level of Src by immunoblotting. G, cells after siRNA transfection were incubated in suspension condition before stimulating with fibronectin/collagen type I (FN/Col.I). H, mock or PIPKIγi2-overexpressing cells incubated in suspension condition were stimulated with 10% FBS or fibronectin/collagen type I to examine the activated Src.

FIGURE 4.

PIPKIγi2 and Src reciprocally regulate their oncogenic function. A–C, siRNA were used to knock down endogenous Src from MDA-MB-231 cells overexpressing PIPKIγi2. Cells were cultured in soft agar for 2–3 weeks before counting the colonies. ConsiRNA, control siRNA. D, PIPKIγi2-overexpressing cells were treated with a pharmacological inhibitor of Src (PP1 or control PP3, 0.5 μm) during suspension culture, and the colonies developed were counted after 2–3 weeks culture in soft agar. All the values are means ± S.D. from three-independent experiments. The error bars represent S.D. (p values are indicated). E, siRNA was used to knock down PIPKIγi2 or PIPKIα from MDA-MB-231 cells transfected with Src. Cells were fixed for IF study to examine the actin cytoskeleton (left panels) and Src localization (right panels). Scale bar, 20 μm. F, cortactin and FAK were immunoprecipitated from mock and Src-infected cells after siRNA transfection for PIPKIγi2 knockdown. Cells were harvested 48 hours post-transfection to immunoprecipitate the endogenous cortactin and FAK followed by immunoblotting using phosphotyrosine antibody. G, siRNA was used for PIPKIγi2 knockdown in MDA-MB-231 cells infected with lentivirus for Src overexpression. Cells were cultured in soft agar for 2 weeks before counting the colonies.

FIGURE 5.

PIPKIγi2 and Src show synergy in inducing oncogenic growth. A and B, PIPKIγi2 and Src were individually expressed or co-expressed into NIH3T3 (A) or MDA-MB-231 cells (B) using lentiviral infection. Their oncogenic growth was monitored by counting colonies developed in soft agar. C, MDA-MB-231 cells overexpressing PIPKIγi2WT or PIPKIγi2KD were infected with lentivirus for Src expression. Oncogenic growth of the infected cells were examined as described above. D and E, the effect of PIPKIγi2 and Src knockdown on oncogenic growth of T47D and HCC1954 cells. All the values are means ± S.D. from three-independent experiments. The error bars represent S.D. (p values are indicated). ConsiRNA, control siRNA.

FIGURE 6.

PIPKIγi2, Src, and talin mutually regulate their targeting to focal adhesions and form a complex in suspension culture. A, upper panels, IF study showing the co-localization of HA-PIPKIγi2 or HA-PIPKIγi1 (green) with endogenous active Src (red) at focal adhesions of MDA-MB-231 cells. Lower panels, MDA-MB-231 cells after siRNA transfection were fixed for IF study to examine the co-localization of active Src (red) with paxillin (green). ConsiRNA, control siRNA. Scale bar, 20 μm. B, the active Src localized at focal adhesions in the cell periphery was counted from at least 30 cells in each experiment. Paxillin was used as a focal adhesion marker. Immunoblot shows the knockdown of indicated proteins in the transfected cells. C, MDA-MB-231 or T47D after siRNA transfection for knockdown of PIPKIγ or PIPKIγi2 were culture in soft agar for 1 week. Colonies developed were processed for IF study to examine the effect on Src (red) and talin (green) localization at plasma membrane. Scale bar, 20 μm. D, MDA-MB-231 cells expressing PIPKIγi1 or PIPKIγi2 were cultured in soft agar for 1 week. The colony developed were processed for IF study to examine the co-localization of PIPKIγi2 or PIPKIγi1 (green) with active Src (red) at plasma membrane. Scale bar, 20 μm. E, HEK293 cells transfected with PIPKIγi1 or PIPKIγi2 or PIPKIγi2 mutant alone or co-transfected with Src were cultured in suspension condition overnight followed by immunoprecipitation (IP) to examine the co-immunoprecipitation of Src and talin with PIPKIγ variants or mutant. F, siRNA was used to knock down talin or FAK from MDA-MB-231 cells overexpressing PIPKIγi2. Their oncogenic growth was monitored by culturing the cells in soft agar as described above. All the values are means ± S.D. from three-independent experiments. The error bars represent S.D. (p values are indicated).

FIGURE 7.

PIPKIγi2 interaction with Src is required for their oncogenic growth control. A, upper panels, endogenous Src was immunoprecipitated (IP) from MDA-MB-231 or MDA-MB-435s cells, and co-immunoprecipitation of PIPKIγ and PIPKIγi2 was examined by immunoblotting. Lower panels, endogenous Src was immunoprecipitated from MDA-MB-231 cells expressing PIPKIγ variants followed by immunoblotting to examine the co-immunoprecipitation of PIPKIγ using anti-HA antibody. B, upper panels, schematic diagram of Src and the domains used for construction of GST fusion proteins. Lower panels, in vitro binding study performed using GST fusion proteins of Src and His-tagged PIPKIγi2 or cell lysates prepared from HA-PIPKIγi2 expressing cells. Bound proteins were examined by immunoblotting. C, HEK293 cells were co-transfected with HA-PIPKIγi2 and Src or its deletion mutants. Co-immunoprecipitation of PIPKIγi2 with Src and its mutant forms was examined by immunoblotting using anti-HA antibody. N-term., N-terminal; C-term., C-terminal. D, MDA-MB-231 cells expressing either PIPKIγi2 or Src or mutant Src alone or co-expressing them were culture in soft agar for 10–12 days before counting the colonies. E, MDA-MB-231 cells co-expressing PIPKIγi2 and Src were infected with lentivirus for the expression of GFP or GFP-C-tail of Src followed by immunoprecipitation of PIPKIγi2 to examine the co-immunoprecipitation of Src. F, these cells were cultured in soft agar for 10–12 days before counting the colonies. All the values are means ± S.D. from three-independent experiments (p values are indicated). NS, nonsignificant p value.

FIGURE 8.

Highly conserved basic amino acids residues in the N terminus of Src are required for oncogenic signaling in synergy with PIPKIγi2. A, schematic diagram depicting the highly conserved basic amino acid residues in N terminus of Src. Mutant forms of the chicken Src used for the study are indicated. N-term., N-terminal; C-term., C-terminal. B, HEK293 cells were co-transfected with PIPKIγi2 and Src or its mutant forms. Cells were harvested 24 hours post-transfection to immunoprecipitate PIPKIγi2. Co-immunoprecipitation of Src was examined by immunoblotting. C, Src or its mutant forms were expressed either individually or with PIPKIγi2 into NIH3T3 cells using lentivirus. 24–48 h post-infection, cells were harvested and cultured in soft agar for 10–12 days followed by counting of the colonies formed. All the values are means ± S.D. from three-independent experiments. The error bars represent S.D. (p values are indicated). NS, nonsignificant p value. D, schematic diagram depicting the collaborative function of PIPKIγi2, Src, and talin in oncogenic growth. PIPKIγi2 simultaneously binds with Src and talin independent of cell adhesion. PIPKIγi2 interaction with Src at its C terminus may help to alleviate intramolecular constraints, promoting Src activation. In turn, this enhances PIPKIγi2 interaction with talin, which promotes PIPKIγi2 and Src to localize at the proximity of integrin and growth factor receptors promoting oncogenic growth signaling.