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An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells
Published on: July 15, 2015
Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity
Juan Carlos Montero1, Ricardo López-Pérez, Jesús F San Miguel
1Centro de Investigación del Cáncer-IBMCC, CSIC-Universidad de Salamanca, and Department of Hematology, University Hospital of Salamanca, Salamanca, Spain.
Background:
c-Kit is expressed in the plasma cells from 30% of patients with multiple myeloma. Two different isoforms of c-Kit, characterized by the presence or absence of the tetrapeptide sequence GNNK in the extracellular domain, have been described. However, their expression and function in myeloma cells are unknown. We explored the function and expression of these c-Kit isoforms in myeloma cells.
Design And Methods:
Expression of c-Kit isoforms was investigated by reverse transcriptase polymerase chain reaction in fresh plasma cells from patients and cell lines. The function of these c-Kit isoforms was analyzed upon expression in myeloma cells. Signaling was investigated by western blotting using antibodies specific for activated forms of several signaling proteins. The impact of c-Kit on the action of drugs commonly used in the treatment of multiple myeloma was investigated by MTT proliferation assays.
Results:
Fresh plasma cells from patients as well as myeloma cell lines expressed the two isoforms of c-Kit. Retroviral infection of myeloma cells with vectors that code for c-Kit-GNNK+ or c-Kit-GNNK- forms demonstrated differences in the kinetics of phosphorylation between these isoforms. Stem cell factor-induced activation of the GNNK- form was faster and more pronounced than that of the GNNK+ form, whose activation, however, lasted for longer. The c-Kit receptors weakly activated the Erk1/2 and Erk5 pathways. Both receptors, however, efficiently coupled to the PI3K/Akt pathway, and stimulated p70S6K activation. The latter was sensitive to the mTOR inhibitor, rapamycin. Studies of drug sensitivity indicated that cells expressing the GNNK- form were more resistant to the anti-myeloma action of bortezomib and melphalan.
Conclusions:
Our data indicate that c-Kit expression in multiple myeloma cells is functional, and coupled to survival pathways that may modulate cell death in response to therapeutic compounds used in the treatment of this disease.
Insights
The two isoforms of c-Kit are functional in multiple myeloma cells, influencing cell survival pathways and drug resistance. This finding suggests c-Kit signaling may impact treatment outcomes for multiple myeloma patients.
Area of Science:
- Oncology
- Molecular Biology
- Cell Signaling
Background:
- c-Kit is expressed in 30% of multiple myeloma plasma cells.
- Two c-Kit isoforms, with and without the GNNK sequence, exist but their role in myeloma is unknown.
Purpose of the Study:
- To investigate the expression and function of c-Kit isoforms in multiple myeloma cells.
- To determine the impact of c-Kit isoforms on cell signaling and drug sensitivity.
Main Methods:
- Reverse transcriptase polymerase chain reaction (RT-PCR) for isoform expression analysis.
- Western blotting to assess signaling pathway activation.
- MTT proliferation assays to evaluate drug sensitivity.
Main Results:
- Both c-Kit isoforms were expressed in patient plasma cells and myeloma cell lines.
- The GNNK- isoform showed faster, more pronounced phosphorylation kinetics than the GNNK+ isoform.
- c-Kit efficiently activated the PI3K/Akt pathway, and the GNNK- isoform conferred resistance to bortezomib and melphalan.
Conclusions:
- c-Kit expression in multiple myeloma is functional.
- Activated c-Kit signaling pathways are linked to cell survival.
- c-Kit may modulate cell death in response to multiple myeloma therapies.
