Convergence of eicosanoid and integrin biology: 12-lipoxygenase seeks a partner

Abstract

Background: Integrins and enzymes of the eicosanoid pathway are both well-established contributors to cancer. However, this is the first report of the interdependence of the two signaling systems. In a screen for proteins that interacted with, and thereby potentially regulated, the human platelet-type 12-lipoxygenase (12-LOX, ALOX12), we identified the integrin β4 (ITGB4).

Methods: Using a cultured mammalian cell model, we have demonstrated that ITGB4 stimulation leads to recruitment of 12-LOX from the cytosol to the membrane where it physically interacts with the integrin to become enzymatically active to produce 12(S)-HETE, a known bioactive lipid metabolite that regulates numerous cancer phenotypes.

Results: The net effect of the interaction was the prevention of cell death in response to starvation. Additionally, regulation of β4-mediated, EGF-stimulated invasion was shown to be dependent on 12-LOX, and downstream Erk signaling in response to ITGB4 activation also required 12-LOX.

Conclusions: This is the first report of an enzyme of the eicosanoid pathway being recruited to and regulated by activated β4 integrin. Integrin β4 has recently been shown to induce expansion of prostate tumor progenitors and there is a strong correlation between stage/grade of prostate cancer and 12-LOX expression. The 12-LOX enzymatic product, 12(S)-HETE, regulates angiogenesis and cell migration in many cancer types. Therefore, disruption of integrin β4-12LOX interaction could reduce the pro-inflammatory oncogenic activity of 12-LOX. This report on the consequences of 12-LOX and ITGB4 interaction sets a precedent for the linkage of integrin and eicosanoid biology through direct protein-protein association.

Fig. 1

P-12-lipoxygenase interacts with integrin β4 subunit in vitro. (a) A431 cells were stimulated with mAb β4 3E1 and harvested at timed intervals from 5–90 min. 12-LOX and the β4 subunit co-immunoprecipitated from untransfected A431 cells (A431), [and A431 cells over expressing 12-LOX (Tx-A431)-Additional file 1]. (b) CHO cells were transfected with different β4 constructs either alone or in combination with 12-LOX: (1) control: vector alone; (2) CHO: nontransfected cells; (3) Txβ4: full-length β4; (4) Txβ4 + 12-LOX: cotransfected with full-length β4 and 12-LOX; (5) Txtruncated1: pCMV-β4 Δ70-660 (c-myc tagged head-less) +12-LOX; (6) Txtruncated2: pCMV-β4 Δ854-1752 (tail-less) +12-LOX. 12-LOX and the β4 subunit coimmunoprecipitated from three transfectants: Txβ4, Txβ4 + 12-LOX, and Txtruncated1 (headless). Positions of 12-LOX and β4 are indicated. The experiment was repeated three times. For each experiment, mouse or rabbit IgG (control 1) and Sepharose 4B-conjugated protein G beads alone (control 2) were used as negative controls. (C) 12-LOX and the β4 subunit co-immunoprecipitated from A431 cells after growth on laminin at the same timed interval as with mAb 3E1 stimulation. Whole cell lysate of A431 stimulated with mAB 3E1 was loaded as a control (A431-3E1T). β4 subunit was detected with mAb 450-11A

Fig. 2

β4 ligation-induced translocation of 12-LOX in A431 cells. A431 cells stimulated with 3E1 mAb for 5, 15, 30, 60 and 90 min. (a) Membrane translocation of 12-LOX from cytosol to membrane. (b) The effect of β4 stimulation on 12-LOX translocation is specific to β4. (c) β1 integrin is detectable in whole cell lysates (WCL), but does not interact with 12-LOX on β4 stimulation

Fig. 3

LC-MS analysis of 12-LOX activity. Following the stimulation with mAb β4 over a time course, each membrane fraction (100,000 × g pellet) was harvested and incubated in DMEM at 37 °C with 10 μM [¹⁴C]-AA for 15 min. Cell lipids were extracted and analyzed as described in Materials and Methods. The data were analyzed by LC-MS in triplicate and error bars represent SEM

Fig. 4

mAb β4 treatment effect on BMD122-induced apoptosis in A431 cells by DNA laddering assay. (a) Comparison of A431 12-LOX transfectants with empty vector control (3.1+). Cells were treated with BMD122 (formerly BHPP) at the concentration indicated for 24 h, low molecular weight DNA was extracted, run on a 1.5 % agarose gel and visualized with ethidium bromide. The middle lanes are DNA markers for comparison. 3.1+: empty vector controls; 12LOX: A431 cells transfected with full-length 12-LOX. (b) A431 cells were pretreated with mAb β4 3E1 (5 μg/ml) before incubation in DMEM in the presence of BMD122, see details in Materials and Methods. Aliquots of DNA extracts were subjected to 1.5 % agarose gel and visualized with ethidium bromide. C: Ethanol as vehicle control. M: DNA marker, left lane

Fig. 5

The α6β4 integrin and 12-LOX activity function in EGF-induced chemotaxis. (a) Migration assay: A431 cells were pretreated with 3E1 antibody for 20 min, then plated on laminin-coated invasion plates either with or without EGF (1 ng/ml) for 3 h in the presence or absence of pharmacological inhibitors as described in Methods. Data represent the mean number of transmigrated cells/microscopic field (± SE). The experiments were repeated three times in triplicate. a, P < 0.01 when compared to no-EGF treated group; b, c, d, P < 0.01 when compared to EGF treated control group. (b) Alternate migration assay: Comparison of chemotaxis of A431 cells toward EGF when stimulated with either mAb 3E1 or laminin in the presence or absence of the 12-LOX specific inhibitor BMD122. (10× magnification) (c) Controls for (b). (d) Absorbance measurements of dye retained by transmigrated cells

Fig. 6

12-LOX knockdown inhibits β4-mediated 12(S)-HETE production, downstream ERK activation, and cellular invasion. (a-c) Validation of 12-LOX KD in A431 cells. (a) 12-LOX mRNA levels measured by RT-PCR in A431 parental, ns (non-silencing) shRNA control cells, and 12-LOX KD cell lines. *p < 0.001 (b) Western blot analysis of 12-LOX protein levels in 12-LOX KD clones, ns shRNA control, parental A431, CHO (negative control for 12-LOX expression; polyclonal platelet-type 12-LOX antibody appears to be recognizing another 12-LOX isoform in CHO cells), PC-3 12-LOX overexpressors along (positive control for 12-LOX), 3.1 empty vector control cells, and platelet lysate (positive control for 12-LOX expression). (c) No increase in 12(S)-HETE levels were seen with 3E1 stimulation in #1 and #2 12-LOX KD clones. 12-LOX activity was measured by 12(S)-HETE production using LC-MS after a 6 h incubation with 3E1 and AA. (d) #1 12-LOX KD cells do not respond to 3E1 stimulation with an increase in phosphorylated ERK levels. Western blot evaluation of phosphorylated ERK with 30 min 3E1 stimulation. Densitometry analysis represents the ratio of phosphorylated ERK to total ERK. (e) #1 12-LOX KD cell invasion is not affected by BMD122 enzymatic inhibition of 12-LOX. Cells were pre-treated with BMD122, then stimulated with 3E1 or EGF and allowed to invade through a Boyden Chamber insert coated with Matrigel for 24 h. Images taken at 10 x. Invaded cells were stained with crystal violet, the dye content dissolved in 10 % acetic acid, and the absorbance measured at OD_570nm. Columns represent the invasion reported as the mean of three samples +/− SE