Development of Herceptin resistance in breast cancer cells
Timothy Kute1, Christopher M Lack, Mark Willingham
1Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1072, USA. tkute@wfubmc.edu
Background:
Herceptin, a humanized antibody to HER-2, is now utilized in the clinic for metastatic breast cancer treatment. The response rate for HER-2+ patients is only 30% and little is known as to mechanisms of resistance. The mechanism of Herceptin action is also unknown but has been related to cell cycle inhibition.
Methods:
The effects of Herceptin and other antibody treatments were determined by cell counting and cell cycle analysis. HER-2 and p27 expression levels were analyzed by flow cytometry and levels of activated AKT were compared by Western blot analysis. Cellular HER-2 and p27 expression was measured by immunofluorescence.
Results:
Herceptin treatment of BT-474 cells results in inhibition of cell growth and arrest in the G1 phase. The efficacy of growth arrest was not directly correlated to the binding affinity of antibodies to Her-2. Our laboratory has developed cell lines that are resistant to Herceptin treatment. In resistant cell lines, binding of antibodies is not hindered. However, Herceptin has completely lost the ability to inhibit cell proliferation. Yet, the mouse isotype 4D5 maintains significant inhibitory activity upon Herceptin-resistant clones.
Conclusions:
Herceptin binds effectively to Her-2 on the cell surface of Herceptin-resistant cell lines and the level of Her-2 expression on the cell surface is not downregulated. Herceptin resistance is not due to downregulation of levels of AKT protein expression, although, phosphorylation of AKT is enhanced in resistant lines and could have a role in resistance. Resistance appears to correlate with the loss of nuclear expression of the cyclin-dependent kinase inhibitor, p27, as defined by immunofluorescence and flow cytometry studies and cdk-2 binding studies.
Insights
Herceptin resistance in breast cancer may stem from a loss of nuclear p27 expression, not HER-2 levels or AKT activity. A related antibody retained efficacy against resistant cells, suggesting alternative therapeutic targets.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Herceptin (trastuzumab) is a HER-2 targeted therapy for metastatic breast cancer.
- Treatment response rates are limited to 30% in HER-2 positive patients.
- Mechanisms of Herceptin resistance and its precise action remain largely unknown.
Purpose of the Study:
- To investigate the mechanisms underlying Herceptin resistance in breast cancer cells.
- To explore the role of HER-2, AKT, and p27 in Herceptin efficacy and resistance.
- To evaluate alternative antibody treatments in Herceptin-resistant models.
Main Methods:
- Cell counting and cell cycle analysis to assess Herceptin's effect on proliferation.
- Flow cytometry and immunofluorescence to measure HER-2 and p27 expression.
- Western blot analysis to compare activated AKT levels.
Main Results:
- Herceptin inhibited cell growth and induced G1 arrest in sensitive cells.
- Herceptin-resistant cell lines showed no hindrance in antibody binding to HER-2.
- A mouse isotype antibody (4D5) retained inhibitory activity against resistant clones.
Conclusions:
- Herceptin resistance is not linked to HER-2 downregulation or AKT pathway alterations.
- Loss of nuclear expression of p27 (a cell cycle inhibitor) correlates with Herceptin resistance.
- Enhanced AKT phosphorylation may contribute to resistance, and alternative antibodies show promise.
Related Concept Videos
02:56Treatment Resistant Cancers
02:57Targeted Cancer Therapies
There are several types of targeted therapies against specific...
02:56Treatment Resistent Cancers
01:25Mechanism of Antibiotic Resistance in MRSA
01:25Clinical Significance of Antibiotic Resistance
01:45Antibiotic Selection