Switch from alphavbeta5 to alphavbeta6 integrin expression protects squamous cell carcinomas from anoikis

Abstract

Stratified squamous epithelia express the alphavbeta5 integrin, but in squamous cell carcinomas (SCCs) there is down-regulation of alphavbeta5 and up-regulation of alphavbeta6. To investigate the significance of this finding, we transduced an alphav-negative human SCC line with retroviral vectors encoding alphav integrins. alphavbeta5-expressing cells underwent suspension-induced apoptosis (anoikis), whereas alphav-negative cells and cells expressing alphavbeta6 did not. Resistance to anoikis correlated with PKB/Akt activation in suspension, but not with changes in PTEN or p110alpha PI3 kinase levels. Anoikis was induced in parental and alphavbeta6-expressing cells by inhibiting PI3 kinase. Conversely, activation of Akt or inhibition of caspases in alphavbeta5-expressing cells suppressed anoikis. Caspase inhibition resulted in increased phosphoAkt, placing caspase activation upstream of decreased Akt activation. Anoikis required the cytoplasmic domain of beta5 and was independent of the death receptor pathway. These results suggest that down-regulation of alphavbeta5 through up-regulation of alphavbeta6 may protect SCCs from anoikis by activating an Akt survival signal.

Figure 1.

Expression of αvβ5 and α4β1 integrins in H357 cells. (a–c) αv expression of H357 retrovirally infected cells (H357αv) compared with parental cells (H357par) and a clone of αv transfected cells, V5, previously described by Jones et al. (1996). (d–f) Infection of H357 cells with α4 alone (H357α4) or in combination with the chick β1 integrin subunit (H357α4β1). (g–l) Comparison of endogenous β1 (g–i) or α6 (j–l) integrin expression in H357par, H357αv, or H357α4. (m–r) H357par and H357αv were labeled with the heterodimer-specific αv antibodies shown. Gray lines, second antibody only; black lines, primary and second antibodies.

Figure 2.

Effects of αvβ5 on cell adhesion, differentiation, and growth in suspension. (a) Adhesion to vitronectin. Diamonds, parental H357 cells; triangles, H357 cells expressing α4β1; squares, H357 cells expressing αvβ5. Data are means and SDs of triplicate samples. (b–g) H357 cells expressing αvβ5 plated on 20 μg/ml vitronectin (b–d and g) or fibronectin (e and f) for 30 min, double labeled with phalloidin (red fluorescence) and antibodies to αvβ5 (b–d), αv (e), or β1 (f and g; green fluorescence). Panel d is the merged image of b and c. Bars, 2 μm. (h) Colony formation in soft agar. 35-mm dishes of parental H357 (H357par), H357 expressing empty retroviral vector (H357pBp), and cells expressing αvβ5 (H357αv). (i–p) Flow cytometric analysis of transglutaminase (B.C1) and involucrin (SY5) expression in H357par, H357αv, or H357α4, and normal primary keratinocytes. Gray lines, second antibody only; black lines, primary and secondary antibodies.

Figure 3.

Expression of αvβ5 in SCC cells induces anoikis. (a) DNA content of H357 cells (parental or transduced with αv or α4) after 0, 24, 48, or 72 h in suspension. Regions corresponding to sub-G1, G1, S, and G2+M are indicated. (b) Percentage of anoikis was determined as the percentage of cells with sub-G1 DNA content. SEM of six experiments are shown, comparing parental H357 cells to those expressing empty vector (pBabe puro), α4, or αv. (c) H357 cells transduced with αv suspended in medium alone (Cont) or supplemented DMSO, z-VAD-fmk (CI) at 100 μM, 100 μg/ml vitronectin (VN), or 100 μg/ml 13C2 anti-αv. Means and SDs of three experiments are shown. (d–g) Flow cytometry profiles of SCC4 and primary human keratinocytes (strain km) labeled with secondary antibody alone (dashed lines) or antibodies to αv or α4 integrins. Gray lines, parental cells; black lines, cells transduced with αv (d and f) or α4 (e and g). (h and i) Percentage of anoikis (sub-G1 DNA) of parental SCC4 cells and primary keratinocytes (km) or cells transduced with αv or α4 integrin subunits. SEM of three experiments are shown.

Figure 4.

Protection from anoikis correlates with Akt activation. (a) Anoikis of H357 cells transduced with αv or α4 integrin after 72 h in suspension. cont, medium alone; DMSO, solvent control; CI, 100 μM z-VAD-fmk; PI3KI, 50 μM LY294002; MEKI, 10 μM UO126; p38MAPKI, 10 μM SB203580. Data are SEM of three experiments. (b) Western blot of H357 cells expressing α4 or αv held in suspension for the number of hours shown. (right hand lane) H357α4 suspended in the presence of LY 294002 for 72 h. Blot was probed with an antibody to phosphoAkt ⁴⁷³ or actin (loading control).

Figure 5.

The proapoptotic effects of αvβ5 are mediated by the β5 cytoplasmic domain. (a and b) Flow cytometry of H357 cells expressing αvβ6 (a) or αvβX6C5 (b), using antibodies specific for the αv heterodimers indicated. (c–e) Western blots of parental H357 cells (par), H357 cells transduced with αv alone (αvβ5) or αv and β6 (αvβ6) probed with antibodies to the integrin subunits shown. Loading controls were actin or Erk MAPK. (f) DNA content of cells suspended for the times indicated. Shown are H357par and cells transduced with αv, αvβ6, or αvβX6C5. (g) SEM of percentage of anoikis in three separate experiments. (h and i) Western blot probed with antibodies to phosphoAkt⁴⁷³, total Akt, or actin. (h) Cells were suspended for the times shown. (right hand lane) Cells suspended with Ly294002 (50 μM). (i) Cells suspended for 72 h.

Figure 6.

Akt activation protects against anoikis. (a) Western blot of adherent H357 cells transduced with M⁺Akt:ER* or A2Akt:ER* alone (parMyr and parA2, respectively) or in combination with αv integrin (αvMyr and αvA2, respectively) and treated with OHT (1 μM) for 1 h. (top) The antibody to phospho-Akt detects endogenous phospho-Akt in all cells (bottom band), and phosphorylated M⁺Akt:ER in cells expressing that construct. (bottom) Probed with anti-ER. (b and c) Anoikis of H357 cells transduced with M⁺Akt:ER* (Myr) or A2Akt:ER* (A2) alone (parent) or in combination with αv or α4 integrins. Cells were suspended for 72 h in the presence of OHT. (c) SEM of three experiments. (d) Western blot of H357αvMyr or αvA2 suspended for 72 h with 0.1 μM OHT, probed with anti-phosphoAkt⁴⁷³ (top), total Akt (middle), and ER (bottom). (top panel) Anti-phosphoAkt recognizes two bands, corresponding to endogenous Akt (bottom band) and to phosphorylated M⁺Akt:ER (top band).

Figure 7.

αvβ5-mediated anoikis is independent of FADD. (a and b) Anoikis of αv-transduced H357 cells suspended in presence of caspase inhibitors z-IETD-fmk (caspase 8), z-LEHD-fmk (caspase 9), and z-VAD-fmk (pan caspase). (b) SEM of three experiments. (c) Flow cytometric profiles of αv-expressing H357 cells before and after transduction with eGFP-dNFADD-AU1. GFP fluorescence is shown. (d) SEM of two experiments comparing the percentage of anoikis after 0 or 72 h in suspension. Parental H357 cells were compared with cells transduced with αv (H357αv) alone or in combination with dnFADD. (e) Effect of z-VAD-fmk (CI) and dnFADD on MDCK anoikis was measured by flow cytometry of annexin V–positive cells. Data are from a single representative experiment.

Figure 8.

Mechanisms by which Akt phosphorylation could be regulated. (a and b) Western blots of parental H357 cells (par) or cells transduced with αv alone or αv and β6 were suspended for 72 h and probed with antibodies to PTEN (a) or PI3 kinase (p110α subunit; b). Actin (a) and Erk MAPK (b) were loading controls. (c) Western blots of H357 cells transduced with αv probed with anti-phosphoAkt⁴⁷³ and total Akt. Cells were suspended for 72 h with 100 μM z-VAD-fmk, z-IETD-fmk, z-LEHD-fmk, or DMSO. Amount of protein loaded per track was greater than sixfold higher than in blots shown in Figs. 4–6.