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. 2000 Sep 18;150(6):1467-78.
doi: 10.1083/jcb.150.6.1467.

A novel function for the tumor suppressor p16(INK4a): induction of anoikis via upregulation of the alpha(5)beta(1) fibronectin receptor

T Plath  1 , K Detjen, M Welzel, Z von Marschall, D Murphy, M Schirner, B Wiedenmann, S Rosewicz
Affiliations

A novel function for the tumor suppressor p16(INK4a): induction of anoikis via upregulation of the alpha(5)beta(1) fibronectin receptor

T Plath et al. J Cell Biol. .

Abstract

The tumor suppressor gene p16(INK4a) inhibits the kinase activity of the cyclin-dependent kinase 4-6/cyclin D complexes and subsequent phosphorylation of critical substrates necessary for transit through the G1 phase of the cell cycle. Recent studies suggested that control of the G1/S boundary might not be the sole biological function of p16(INK4a). We hypothesized that p16(INK4a) might influence hitherto unknown critical features of a malignant epithelial phenotype, such as anchorage dependence. Here we provide evidence that stable transfection of p16(INK4a) restitutes apoptosis induction upon loss of anchorage (anoikis) in a variety of human cancer cells. Anoikis in p16(INK4a)-transfected cells was evidenced by DNA fragmentation and poly(ADP-ribose) polymerase cleavage upon cultivation on polyhydroxyethylmethacrylate-coated dishes and was associated with suppression of anchorage-independent growth as well as complete loss of tumorigenicity. p16(INK4a)-mediated anoikis was due to selective transcriptional upregulation of the alpha(5) integrin chain of the alpha(5)beta(1) fibronectin receptor as detected by FACS((R)) analysis, immunoprecipitation, Northern blotting, and nuclear run-on assays. Addition of soluble fibronectin and inhibitory alpha(5) antibodies to nonadherent cells completely abolished p16(INK4a)-mediated anoikis, whereas laminin was ineffective. Furthermore, antisense-induced downregulation of the alpha(5) integrin chain in p16(INK4a)-transfected cells restored resistance to anoikis. These data suggest a novel functional interference between a cell cycle-regulating tumor suppressor gene and membrane-bound integrins, thus regulating a hallmark feature of an epithelial transformed phenotype: susceptibility to anoikis.

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Figures

Figure 1
Figure 1
p16INK4a inhibits anchorage-independent growth. (A) Human full-length p16INK4a cDNA was cloned into the eukaryotic expression vector pRC/CMV and stably transfected in Capan-1 cells. Three randomly selected p16INK4a-positive clones (p16/1,2,3) were chosen for further experiments. Cdk4 was immunoprecipitated from whole cell lysates of either control or p16INK4-expressing clones. Immunoprecipitates were then analyzed by Western blotting using monospecific antibodies against p16INK4a and Cdk4. (B) Anchorage-independent growth was examined using a soft agar colony formation assay. 103 cells were seeded in a methylcellulose/agar mixture and incubated for 12 d. Vital colonies of >20 cells were counted using a microscope. Shown are the mean ± SEM of three independent experiments, each performed in triplicates.
Figure 2
Figure 2
Tumorigenicity in vivo. (A) Nude mice were subcutaneously injected with 2 × 106 Capan-1 cells from either control clones (•, wild-type; ▴, mock 1; and ▾, mock 2) or p16INK4-transfected clones (○, p16/1; ▿, p16/2; and ▵, p16/3). Each clone was injected in at least six animals. Tumor growth was then monitored at the indicated time points. Shown is a representative (mean ± SEM) of two independent experiments yielding identical results. (B) In situ apoptosis detection. Tissue sections of xenografts were prepared at day 2 after cell injection. Cellular apoptosis was detected using the TUNEL assay as described in Materials and Methods. Arrows indicate representative apoptotic cells characterized by a brown color (magnification, ×100).
Figure 3
Figure 3
Analysis of anoikis. Anoikis was examined by three different experimental procedures. For all experiments, 5 × 105 resuspended cells were plated on polyHEMA-coated dishes. After 20 h, cells were harvested and subsequently analyzed. A representative of three independent experiments yielding identical results is shown. (A) FACS® analysis. Cells were ethanol-fixed, stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells containing subdiploid DNA is indicated in each panel. (B) PARP cleavage. Cell lysates were prepared and immunoblotted with an mAb to PARP. The indicated molecular mass of the full-length PARP protein and its apoptotic cleavage fragment were deduced from a molecular size marker electrophoresed in parallel. (C) DNA laddering. From each clone, genomic DNA was extracted and 1 μg was electrophoresed on a 1% agarose gel. Fragment sizes of the 100-bp marker are indicated.
Figure 4
Figure 4
p16INK4a induces anoikis in cell lines from different origin. (A) p16INK4a was stably transfected into pancreatic tumor cell line Capan-1, melanoma cell line NKI4, hepatocellular carcinoma cell line SKHep1, and kidney cell line MDCK. Expression of p16INK4a was confirmed by Western blotting using monospecific p16INK4a antibodies. (B) For determination of anoikis, 5 × 105 cells of each control or p16INK4a-positive clone were plated on polyHEMA-coated dishes and incubated for 6 h (MDCK), 20 h (Capan-1 and SKHep1), or 72 h (NKI4). The amount of apoptotic cells was subsequently determined by FACS® analysis. Shown are the mean ± SEM of three independent experiments, each performed in triplicates.
Figure 5
Figure 5
Analysis of integrin expression. (A) Capan-1 cells were incubated with the indicated mAb against various α and β integrin subunits and subsequently analyzed by flow cytometry. Results from three control (wild-type, mock 1/2) and three p16 (1/2/3) clones are summarized and the mean ± SEM of two independent experiments is shown. (B) Biotinylated cell surface proteins were immunoprecipitated with an α5 integrin mAb and detected using a streptavidin-peroxidase conjugate in ECL visualization. The size of α5 and β1 integrin subunits was deduced from markers electrophoresed in parallel. (C) Poly A+ purified mRNA (5 μg/lane) of the indicated cell clones was analyzed by Northern blotting using a [32P]dCTP-labeled α5 integrin cDNA probe. Bands reflecting α5 integrin mRNA (4.2 kb) and rRNAs are indicated. As loading control, samples were stained with ethidium bromide before blotting (bottom). (D) Nuclei were prepared from a mock-transfected and a p16INK4a-positive clone. After in vitro transcription in the presence of [α-32P]UTP, 32P-labeled RNA (5 × 106 cpm) was hybridized for 48 h to cDNAs of p16INK4a, GAPDH, and α5 integrin subunit immobilized on a nitrocellulose membrane. No hybridization signal was detectable when α-amanitin was added to the transcription reaction, confirming the specificity of the observed hybridization signals (not shown). One representative autoradiograph out of three independent experiments yielding identical results is shown.
Figure 6
Figure 6
Effects of fibronectin on anoikis. (A) 5 × 105 cells were plated on polyHEMA dishes and incubated for 20 h with soluble fibronectin (0.3 mg/ml) or laminin (0.15 mg/ml). Apoptotic cells were subsequently quantitated by flow cytometry. Shown is the mean ± SEM of three independent experiments. (B) 5 × 105 cells of Capan-1 clone p16/1 were plated on polyHEMA dishes and incubated with the indicated concentrations of soluble fibronectin and inhibitory anti-α5 antibody for 20 h. Apoptosis was detected and quantitated by flow cytometry measuring the apoptotic pre-G1 peaks. One representative of three experiments is shown.
Figure 7
Figure 7
Antisense-induced downregulation of the α5 integrin chain. 1.5 × 107 cells of Capan-1 clone p16/1 and p16/2 were cotransfected using lipofectamine with the vectors pCMV-5′ α5/antisense and pIRES1-hyg. After 4 d of selection in the presence of 0.1 mg/ml hygromycin, cells were pooled and α5 expression was detected by FACS® analysis using a monospecific antibody against α5 integrin chain and immunoglobulins (Ig) as isotype control. Demonstrated are the α5 expression levels of Capan-1 wild-type (wt), p16INK4a-transfected (p16/1), and p16INK4a plus α5 antisense cotransfected (p16/α5 as) Capan-1 clones. A representative out of three experiments yielding identical results is shown.
Figure 8
Figure 8
Effects of α5 integrin downregulation on p16INK4a-mediated anoikis. 5 × 105 cells of control, p16INK4a-positive, or p16INK4a-positive plus α5 antisense–transfected clones generated as described in the legend to Fig. 7, were plated on polyHEMA-coated dishes for 20 h and subsequently analyzed for cell cycle distribution by flow cytometry. Shown is a representative cell cycle distribution (A) and the mean ± SEM of three independent experiments (B). (C) PARP cleavage. After cultivation on polyHEMA, cell lysates were prepared and immunoblotted with an mAb against PARP.
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