PTHrP Overexpression Increases Sensitivity of Breast Cancer Cells to Apo2L/TRAIL

Abstract

Parathyroid hormone-related protein (PTHrP) is a key component in breast development and breast tumour biology. PTHrP has been discovered as a causative agent of hypercalcaemia of malignancy and is also one of the main factors implicated in breast cancer mediated osteolysis. Clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis. Furthermore, PTHrP has been demonstrated to cause tumour cell death both in vitro and in vivo. Apo2L/TRAIL is a promising new anti-cancer agent, due to its ability to selectively induce apoptosis in cancer cells whilst sparing most normal cells. However, some cancer cells are resistant to Apo2L/TRAIL-induced apoptosis thus limiting its therapeutic efficacy. The effects of PTHrP on cell death signalling pathways initiated by Apo2L/TRAIL were investigated in breast cancer cells. Expression of PTHrP in Apo2L/TRAIL resistant cell line MCF-7 sensitised these cells to Apo2L/TRAIL-induced apoptosis. The actions of PTHrP resulted from intracellular effects, since exogenous treatment of PTHrP had no effect on Apo2L/TRAIL-induced apoptosis. Apo2L/TRAIL-induced apoptosis in PTHrP expressing cells occurred through the activation of caspase-10 resulting in caspase-9 activation and induction of apoptosis through the effector caspases, caspase-6 and -7. PTHrP increased cell surface expression of Apo2L/TRAIL death receptors, TRAIL-R1 and TRAIL-R2. Antagonistic antibodies against the death receptors demonstrated that Apo2L/TRAIL mediated its apoptotic signals through activation of the TRAIL-R2 in PTHrP expressing breast cancer cells. These studies reveal a novel role for PTHrP with Apo2L/TRAIL that maybe important for future diagnosis and treatment of breast cancer.

Figure 1. PTHrP increases growth of breast cancer cells.

Cells (6×10⁴ per well) were seeded in triplicate into 6 well plates and allowed to adhere for 24 h. Cells were serum starved for 24 h then media was replaced with full serum media. Cell growth was determined by collecting cells every 24 h for a total of 7 days and counted using a Coulter counter. Data is a representative of three independent experiments, the mean ± SD of 6 replicates are provided. ***p<0.001 relative to parental or vector control (VC) cells.

Figure 2. PTHrP increases cell cycle progression of breast cancer cells.

Cells were serum starved for 24 h then media was replaced with full serum media. Cells (A, MCF-7 and B, MCF-7+PTHrP) were fixed in 70% ethanol, washed then stained with a solution containing 40 µg/ml propidium iodide and analysed by flow cytometric analysis. Data presented is a representative of three independent experiments.

Figure 3. PTHrP sensitises breast cancer cells to Apo2L/TRAIL induced cell death.

A) MCF-7 and MCF-7+PTHrP cells (1×10⁴ cells per well) were seeded in quadruplicate into 96 well plates and treated with Apo2L/TRAIL or untreated for 24 h. Cell viability was determined by crystal violet staining and quantified by OD₅₇₀ measurement using a spectrophotometer. Data are presented as the mean ± SD of three independent experiments and are expressed as a percentage of the untreated control cells. B) MDA-MB-231 cells were cultured as above and treated with various portions of PTHrP (10⁻⁸ M) for 12 h, followed by treatment with Apo2L/TRAIL for a further 12 h. Cell viability was assessed as above. ***p<0.001 relative to untreated controls.

Figure 4. Apo2L/TRAIL induces apoptosis in PTHrP overexpressing breast cancer cell through activation of the intracellular apoptotic pathway.

A) Cells were seeded onto chamber slides and treated with Apo2L/TRAIL (TX) or untreated (UT) for 24 h. Cells were fixed with methanol and incubated with DAPI, before washing in PBS and mounting with PBS/glycerine. DAPI staining was visualised by fluorescence microscopy. B) Apoptosis was assessed by DNA laddering assay. Cells were cultured and treated with Apo2L/TRAIL (TX) or untreated (UT) for 24 h. DNA was isolated and treated using the Apoptotic DNA Laddering Kit (Roche). C) Caspase expression. Cells were cultured as indicated above and lysed after 24 h. Cell extracts were analysed by Bis-Tris gel electrophoresis and transferred to PVDF membranes. Caspase pre-cursors-6, -7, -8, -9, -10, PARP and β-actin protein levels was assessed by immunoblotting.

Figure 5. The cell surface expression of TRAIL-R1 and TRAIL-R2 is induced in PTHrP overexpressing cells.

Cells (A, MCF-7 and B, MCF-7+PTHrP) were cultured, washed in PBS and blocked in 10% BSA/PBS +0.1% Azide. Monoclonal antibodies against specific for the human Apo2L/TRAIL death and decoy receptors were added to the cells, and were detected by PE-conjugated secondary antibodies. Cells were washed in PBS and fixed in 1% paraformaldehyde before detection of receptors by flow cytometric analysis. Data presented is a representative of three independent experiments.

Figure 6. Apo2L/TRAIL induced apoptosis in PTHrP overexpressing breast cancer cells is signalled through TRAIL-R1.

Cells were cultured and incubated with polyclonal antibodies against A) TRAIL-R1 and TRAIL-R2, B) TRAIL-R1 only and C) TRAIL-R2 only for 24 h, then treated with Apo2L/TRAIL for a further 24 h. Cell viability was assessed by crystal violet staining and quantified by OD₅₇₀ measurement using a spectrophotometer. Data are presented as the mean ± SD of three independent experiments and are expressed as a percentage of the untreated control cells, *p<0.05, **p<0.01, ***p<0.001.

Figure 7. Schematic Diagram of Proposed Model of Relationship Between PTHrP and Apo2L/TRAIL in Breast Cancer Cells.

Depicted is a model for PTHrP overexpression and Apo2L/TRAIL induced apoptosis in breast cancer cells. In PTHrP cells preferential binding of Apo2L/TRAIL ligand to TRAIL-R2 drives the activation of caspase-10. This leads to the activation of caspase-9, 6 and 7 and resulting in apoptosis. The actions of PTHrP were not through PTH1R signalling (1), but possibly through actions of internalised PTHrP (2), or intracellular PTHrP (3) or nuclear PTHrP (4): internalised PTHrP via PTH1R (2) is unlikely since exogenous treatment of cells with PTHrP do not result in apoptosis. Nuclear PTHrP can affect cell cycle and caspases to regulate apoptosis. Dotted line indicates indirect and/or unknown interaction and solid lines indicates direct interaction.