Pdcd4 repression of lysyl oxidase inhibits hypoxia-induced breast cancer cell invasion

Abstract

Metastasis to bone, liver and lungs is the primary cause of death in breast cancer patients. Our studies have revealed that the novel tumor suppressor Pdcd4 inhibits breast cancer cell migration and invasion in vitro. Loss of Pdcd4 in human nonmetastatic breast cancer cells increased the expression of lysyl oxidase (LOX) mRNA. LOX is a hypoxia-inducible amine oxidase, the activity of which enhances breast cancer cell invasion in vitro and in vivo. Specific inhibition of LOX activity by beta-aminopropionitrile or small interfering RNA decreased the invasiveness of T47D and MCF7 breast cancer cells attenuated for Pdcd4 function. Most significantly, loss of Pdcd4 augments hypoxia induction of LOX as well. Conversely, overexpression of Pdcd4 significantly reversed the hypoxia induction of LOX expression in T47D cells attenuated for Pdcd4. However, Pdcd4 did not affect hypoxia-inducible factor-1 (HIF-1) protein expression or HIF-1-responsive element-luciferase activity in response to hypoxia, suggesting that Pdcd4 regulation of LOX occurs through an HIF-independent mechanism. Nevertheless, the loss of Pdcd4 early in cancer progression may have an important role in the increased sensitivity of cancer cells to hypoxia through increased LOX activity and concomitant enhanced invasiveness.

Figure 1

Pdcd4 inhibits breast cancer cell migration and invasion. (a) Pdcd4 protein depletion on short hairpin RNA (shRNA)-mediated knockdown in T47D cells (pooled) and independent subclones (right panel) was verified by western blot. (b) Pdcd4 depletion enhanced migration and invasion of T47D cells. (c) Pdcd4 protein depletion on shRNA-mediated knockdown in independent subclones of MCF7 cells also resulted in increased matrigel invasion (d).

Figure 2

Pdcd4 knockdown results in an increase in LOX expression. (a) Western blot with anti-LOX antibody reveals that levels of the LOX pre-proprotein (50 kDa) increase on downregulation of Pdcd4 protein levels in the poorly invasive T47D breast cancer cell line. Relative intensity was calculated using ImageJ and expressed as fold change normalized to the intensity of each protein in lane 1 (T47D parental cell line). Quantitative RT–PCR analysis of LOX mRNA expression in T47D (b) and MCF7 (c) cells transfected with siRNA to Pdcd4, using LOX-specific PCR primers. Expression levels were normalized to 18 s RNA expression. Results were from three independent experiments and plotted as fold expression relative to LOX expression (± s.d.) in the control cell line arbitrarily set at 1.0.

Figure 3

Inhibition of LOX activity suppresses migration and invasion in cells attenuated for Pdcd4. (a) Specific inhibition of LOX activity with treatment of LOX inhibitor βAPN (0, 100 or 300 µm) results in decreased migration (b) and matrigel invasion (a, c) of T47D cells attenuated for Pdcd4 function (T47DshPdcd4). The highly invasive and metastatic cell line MDA-MB-231 was used as control. All results were from three independent experiments (n = 6 each) and results were expressed as percentage of control (no βAPN treatment) for each cell line. Statistical significance (*) was established by analysis of variance (P < 0.05).

Figure 4

Pdcd4 inhibition of LOX suppresses hypoxia-induced breast cancer cell invasion. Migration (a) and matrigel invasiveness (b) of the parental, T47DshPdcd4 and control T47Dshscram cell lines were monitored under normoxic (21% O₂) and hypoxic (1% O₂) growth conditions. The invasive cell line MDA-MB-231 was used as a positive control. We determined the average number of migratory or invasive cells in three independent experiments and the results are presented as a ratio of the mean number of cells migrating or invading in hypoxia versus normoxia, that is, [Migration^hyp/Migration^norm] (a) and [Invasion^hyp/Invasion^norm] (b). Knockdown of LOX reversed the invasive phenotype of T47DshPdcd4 cells in the matrigel invasion assay. Small interfering RNA to LOX was used to knockdown LOX expression in the T47DshPdcd4 stable cell line. Quantitative PCR analysis of LOX expression was monitored to confirm the knockdown (c). Matrigel invasion assay was performed on control (T47Dshscram) and T47DshPdcd4 cells under both normoxic and hypoxic growth conditions (d).

Figure 5

Hypoxia enhances expression of LOX mRNA and protein in cell lines lacking Pdcd4. Cells were grown under normoxic and hypoxic growth environments. (a) Total RNA was isolated for quantitative reverse transcription (QRT–PCR) analysis to assay LOX gene expression. (b) Total cell lysate was analyzed by SDS–polyacrylamide gel electrophoresis and immunoblotted with anti-Pdcd4 and anti-LOX antibodies. β-Actin expression was monitored as loading control and HIF-1α levels were monitored to ensure hypoxia response. Relative intensity was calculated using ImageJ and expressed as fold change normalized to intensity of each protein in lane 1 (T47D parental cells under normoxia). (c) AKT stable overexpression downregulates Pdcd4 protein. Western blot was performed on whole-cell extracts from T47D, T47Dmyr-AKT and T47DpFBneo control cells grown under normoxia and hypoxia. Rapamycin (20 nm) treatment inhibits AKT-mediated degradation of Pdcd4. (d) Loss of Pdcd4 in T47DMyrAKT results in increased hypoxia-induced LOX expression in T47DmyrAKT cells. (e) Western blot of whole-cell extracts after transient overexpression of degradation-resistant (TM) but not wild-type (WT) Pdcd4 restored Pdcd4 protein levels in T47DmyrAKT cells. (f) Overexpression of Pdcd4 reverses hypoxia-induced LOX expression in cells attenuated for Pdcd4. QRT–PCR analysis of total RNA from Pdcd4-attenuated cells demonstrates that overexpression of degradation-resistant Pdcd4 significantly reverses the elevation of hypoxia-induced LOX expression (mean values ± s.d shown). Results for all quantitative PCR analyses were from three independent experiments. 18 s RNA expression was measured for normalization and results were expressed as fold relative LOX expression in the cell lines indicated. Statistical significance was established by Student’s t-test analysis (P < 0.05).

Figure 6

Pdcd4 regulates LOX expression by an HIF-1-independent mechanism. Hypoxia response was monitored by expression of HRE-luciferase reporter transfected into T47D parental, shscram and shPdcd4 cell lines grown under normoxic and hypoxic conditions (a). Response to TPA treatment was monitored using the AP-1-luciferase reporter transiently transfected into the above cell lines (b). Cells were then treated for 18 h with dimethylsulfoxide or TPA. Results were from three independent transfections (n = 9). Proposed model for the role of Pdcd4 in hypoxia induction of LOX (c). Pdcd4 suppresses breast cancer cell invasion by inhibiting the expression of LOX (i). Pdcd4 depletion enhances LOX expression and LOX activity-dependent invasive potential of T47D cells under normoxia (ii). Loss of Pdcd4 increases sensitivity to hypoxia-induced breast cancer cell migration, invasion and, possibly, metastasis through HRE-independent regulation of LOX expression (iii).