Cell aggregation induces phosphorylation of PECAM-1 and Pyk2 and promotes tumor cell anchorage-independent growth

Kidney epithelial cells (MDCK), human lung adenocarcinoma cells (NCI-H1792), human non-small cell lung cancer cells (SK-LU-1), human lung adenocarcinoma cells (A549), and large cell human lung carcinoma cells (NCI-H460) were obtained from American Type Cell Culture Collection (ATCC, Rockville, MD). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 IU·ml^-1 streptomycin, and 100 μg·ml^-1 penicillin. HBE4-E6/E7 (human papillomavirus 16E6/E7 transformed normal lung bronchus epithelial cells), obtained from ATCC, were cultured in Keratinocyte Serum-Free Medium (GIBCO, USA) with 0.05 mg·ml^-1 bovine pituitary extract, 5 ng·ml^-1 recombinant human epithelial growth factor, and 10 ng·ml^-1 cholera toxin.

Cells were suspended in 0.3% agar medium (DMEM containing 10% FBS) and then plated on a 0.6% agar base layer at a concentration of 4 × 10⁴ cells per 60-mm dish. The cells were incubated in a humidified atmosphere (5% CO₂) at 37°C. The number of colonies that were 50 μm or larger were counted after two weeks.

Cells were grown to 70% confluence and then trypsinized. They were then seeded in 60-mm polyHEMA-coated petri dishes at different densities and incubated for different periods of time. The cells were subsequently examined under an inverted phase-contrast microscope, photographed, and counted. The polyHEMA-coated dishes were prepared by coating the plates twice with 2 ml polyHEMA solution [10 mg/ml polyhydroxyethylmethacrylate (Aldrich Chemical Co., Milwaukee, WI) in ethanol] and were dried in a tissue culture hood for 24 h. They were then washed twice with PBS before use.

Cells were lysed in DNA lysis buffer containing 0.5% Triton X-100, 10 mM EDTA (pH 8.0) and 10 mM Tris (pH7.6). The cell lysates were kept on ice for 1 h and then extracted twice with an equal volume of chloroform/phenol, followed by one extraction with chloroform. DNA was precipitated, washed with 70% ethanol, air-dried at room temperature, and resuspended in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 7.5). DNA was resolved by electrophoresis in 2% agarose and photographed.

Apoptosis was measured with the Annexin V-FITC Apoptosis Detection KIT (CALBIOCHEM) and analyzed by flow cytometry (FACScan; Becton Dickinson).

Cells were washed with PBS and lysed in Triton lysis buffer (2% Triton X-100, 10 mM EGTA, 15 mM HEPES, 145 mM NaCl, 0.1 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 10 mg·ml^-1 aprotinin, 10 mg·ml^-1 leupeptin, and 2 mM sodium orthovanadate, pH 7.4) at 4°C for 30 min. Triton-soluble and -insoluble fractions were separated by centrifugation at 15,000 × g for 5 min at 4°C. The supernatant was precleared by incubating with 40 μl of protein A-agarose beads for 30 min at 4°C and then centrifuged. Precleared lysates were incubated overnight with PECAM-1 or Pyk2 antibody, and the immune complexes were recovered by incubating with 40 μl of protein-A agarose beads and centrifuging. The immunoprecipitates were washed three times with the Triton lysis buffer and then resolved by 7.5% SDS-PAGE.

pRETRO-SUPER RNA interference constructs were created as described previously [40]. The oligonucleotide sense strand for PYK2 was 5'-gatccccCTGGTCAAATGCACTGTCCttcaagagaGGACAGTGCATTTGACCAG tttttggaaa3', and for PECAM was 5'-gatcccc ACCACTGCAGAGTACCAGCttcaagagaGCTGGTACTCTGCAGTGGT tttttggaaa3'. The 19-nt target sequences are indicated in capital letters.

Ecotropic retroviral supernatants were produced by transfection of packaging cells using calcium-phosphate precipitation method. At 48 h post-transfection, the cell culture medium was filtered through a 0.45 μm filter, 4 μg/ml polybrene was added, and the virus-containing supernatant was used to infect H460 cells. After 6 h of infection, cells were allowed to recover for 24 h in fresh medium. Infected cells were selected with puromycin (2 μg/ml) (Invitrogen, Carlsbad, CA) for 48 h.

293T cells (1 × 10⁶) were transfected with pcDNA 3.1 empty vector, PECAM FL construct, PECAM Mu (Δ11-16) construct, PYK2 construct, or a combination of these using Lipofectamine Reagent (Invitrogen, Carlsbad, California). The pcDNA/Pyk2 construct was a kind gift from Dr. Mike Schaller (UNC-Chapel Hill). The full-length PECAM-1 and Δ11-16 PECAM-1 (exons 11-16 deleted) constructs were a kind gift from Dr. Steven M. Albelda (Univ. Pennsylvania). After 48 h, cell lysates were prepared in lysis buffer (100 mM Tris, pH 8.0, 100 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) and precleared with protein A-Sepharose beads (Amersham Biosciences). Protein lysates (1-mg aliquots) were immunoprecipitated with anti-PECAM-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Pyk2 antibody (Biosource), followed by immunoblotting with an anti-Pyk2 antibody and anti-PECAM-1 antibody. Immunoreactivity was detected using the Enhanced Chemiluminescence system (ECL, Amersham Pharmacia Biotech, Piscataway. NJ).

Cells were washed with PBS and resuspended in lysis buffer (50 mM Tris PH 7.5, 150 mM NaCl, 1%NP40, 0.1%SDS, 1 mM PMSF, 1 mM orthovanadate, 1 mM Na4P2O7,10 mg·ml^-1 aprotinin, and 10 mg·ml^-1 leupeptin ) at 4°C for 30 min and centrifuged (13,000 × g, 5 min at 4°C). Protein concentrations of the lysates were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, CA ). Protein (30 μg) was resolved on SDS-PAGE and transferred onto a nitrocellulose filter. Blocking was carried out for 1 h at room temperature in 5% nonfat milk made in TBS containing 0.05% Tween 20 (TBST). The membrane was then incubated overnight at 4°C in primary antibody (1:1000 dilution) followed by a 1-h incubation with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (1:2000 dilution). The 4G10 (anti-phosphotyrosine) antibody was purchased from Millipore, anti-phosphorylated-Pyk2 (pY-881) was purchased from Biosource (Invitrogen), and anti-cleaved caspase-3 (Asp175) antibody was purchased from Cell Signaling. Immunoreactivity was detected using the Enhanced Chemiluminescence system (Amersham).

We used several cell culture-based approaches to study the molecular mechanism of tumor cell anoikis resistance, or anchorage-independent survival. Our first strategy was to culture cells in polyHEMA-coated dishes, which prevents cells from attaching to the culture dish and forces the cells to grow in suspension [41, 42]. We observed that these tumor cells often formed cell aggregates, and the size and tightness of the aggregates varied depending on the cell line used (Fig. 1A upper). The H1792 and H460 cells formed large and compact aggregates, while the SK-LU-1 and A549 cells formed small and loose aggregates. Most of the MDCK and HBE cells, the non-tumor cell lines, did not form aggregates (Fig. 1A upper).

We analyzed the survival and growth of all of our cell lines in suspension and in soft agar to determine whether cell aggregation played a role in their survival and growth. We found that cell aggregation correlated with soft agar growth (Fig. 1A and 1B) (r = 0.999, P < 0.001). Normal MDCK and HBE cells, which did not form aggregates, failed to form colonies in soft agar. The SK-LU-1 cells, which did not form large aggregates in polyHEMA suspension, and the A549 cells, which formed loose aggregates, grew more slowly in soft agar than the H1792 and H460 cells, which formed large, tight aggregates and grew faster in soft agar (Fig. 1A upper and lower). In suspension culture we also found that tumor cells that formed large aggregates, such as H460 and H1792 cells, grew faster than those cells that formed small aggregates, such as SK-LU-1 and A549 cells (Fig. 1C). These results clearly demonstrate that the ability of the tumor cells to form aggregates in polyHEMA suspension not only correlates with the tumor cell growth in polyHEMA suspension but also correlates with their ability to grow in soft agar.

In order to further examine the role of cell aggregation in cell growth, we used methyl cellulose in the culture medium to decrease cell aggregation in suspension. The size of cell aggregates in the H1792 and H460 decreased in the methyl cellulose suspension (MS) cultures compared with that in the polyHEMA suspension (S) cultures. We observed decreased cell growth in the H1792 and H460 cells in methyl cellulose cultures compared to polyHEMA suspension cultures (Fig. 1D). These data suggest that cell aggregation provides growth signals to tumor cells in suspension.

However, we did not observe a correlation between cell aggregation and survival in the case of tumor cells in suspension. The H460 tumor cells, which formed large and tight aggregates in suspension, underwent partial anoikis in suspension (Fig. 2A-C). On the other hand, the SK-LU-1 tumor cells, which did not form large aggregates and grew slowly in suspension (Fig. 1C), were completely resistant to anoikis (Fig. 2A). While aggregation of H460 cells provided adequate signals for cell growth (Fig. 1C), it was not sufficient to protect the cells from undergoing anoikis (Fig. 2A). These data suggest that the signals for tumor cell anchorage-independent survival and growth were distinct.

In order to understand the molecular nature of the cell aggregation-generated survival and growth signals, we analyzed the phosphorylation/activation of PECAM-1 and Pyk2. PECAM protein was immunoprecipitated with PECAM antibody and then immunoblotted with 4G10 antibody (anti-phosphotyrosine monoclonal antibody) to detect tyrosine phosphorylation. Interestingly, we found that the phosphorylation/activation of PECAM-1 and Pyk2 correlated with cell aggregation, although the total protein level of PECAM-1 and Pyk2 were not related to cell aggregation (Fig. 3A, B and Additional file 1 Figure S1). High- and low-density suspension cultures were established in order to study the phosphorylation status of these molecules in relation to cell aggregation. H460, SK-LU-1, and HBE cells formed larger aggregates in high-density cultures compared to low-density cultures. H460 and H1792 formed larger aggregates in polyHEMA cultures compared to methyl cellulose cultures. We observed higher levels of phosphorylated PECAM-1 and Pyk2 in large aggregates (high-density cultures, H) than in small aggregates or single cells (low-density cultures, L) (Fig. 3A)Similarly, higher levels of phosphorylated PECAM-1 and Pyk2 were seen when cells were cultured in large aggregates (polyHEMA suspension culture, S) compared to small aggregates (methyl cellulose suspension culture, MS) (Fig. 3B), suggesting that the phosphorylation/activation of PECAM-1 and Pyk2 correlated with cell aggregation.

The above experiments suggested that the phosphorylation/activatio of PECAM-1 and Pyk2 correlated with cell aggregation. In order to investigate the role of PECAM-1 and Pyk2 in the formation of cell aggregates, we used RNA-interference (RNAi) technology to knock down the expression of PECAM-1 or Pyk2 in NCI-H460 and A549 cells. We found significantly decreased expression levels of the target proteins in the RNAi-infected cells (Fig. 4A).

Cells with knock-down of Pyk2 or PECAM-1 expression showed a decrease in the size and tightness of cell aggregates (Fig. 4B) along with a decreased ability to grow in soft agar (Fig. 4C). PECAM or Pyk2 knock-down cells also formed only about 30% to 50% the number of colonies in soft agar as the control cells (Fig. 4D). Taken together, these data demonstrate that the expression of Pyk2 and PECAM is important in the formation of tumor cell aggregates and could promote the anchorage-independent growth of H460 and A549 cells in soft agar.

Since both Pyk2 and PECAM-1 are important in mediating the formation of tumor cell aggregates and promoting the anchorage-independent growth, we used co-immunoprecipitation to investigate possible interactions between PECAM-1 and Pyk2. We found that PECAM-1 and Pyk2 co-immunoprecipitated in H460 and A549 cells, suggesting an endogenous interaction between these two molecules in these cells (Fig. 5A and 5B).

To further confirm the interaction between Pyk2 and PECAM-1, we co-transfected 293T cells with an expression vector for Pyk2, along with an expression vector for full-length PECAM-1 or PECAM-1 carrying an exon 11-16 deletion. We demonstrated that Pyk2 was co-immunoprecipitated with the full-length version, but not the deletion mutant, of PECAM-1 (Fig. 5C) clearly demonstrating a direct physical interaction between PECAM-1 and Pyk2.