Suberoylanilide Hydroxamic Acid Overcomes Erlotinib-Acquired Resistance via PTEN-Mediated Apoptosis in Non-Small Cell Lung Cancer
Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality globally, with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) like erlotinib serving as cornerstone therapies for EGFR-mutated cases. However, acquired resistance to EGFR-TKIs inevitably develops, limiting their long-term efficacy. This study investigates the role of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) in overcoming erlotinib resistance, focusing on the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) as a critical mediator of this process.
Establishment of Erlotinib-Resistant NSCLC Cells
To model acquired resistance, the erlotinib-sensitive human NSCLC cell line PC-9 was chronically exposed to progressively increasing concentrations of erlotinib, resulting in the derivative PC-9/ER cell line. Resistance was quantified using the half-maximal inhibitory concentration (IC50) values: parental PC-9 cells exhibited an IC50 of 0.029 μmol/L for erlotinib, while PC-9/ER cells demonstrated a 74.48-fold increase in resistance (IC50 = 2.16 ± 0.21 μmol/L). The H1975 cell line, harboring the EGFR T790M resistance mutation, showed comparable resistance (IC50 = 2.38 ± 0.87 μmol/L), validating the model.
SAHA Synergizes with Erlotinib to Inhibit Resistant Cell Proliferation
The combination of SAHA and erlotinib exhibited synergistic effects on erlotinib-resistant cells. Cell viability assays revealed that SAHA (0.25–4 μmol/L) or erlotinib (0–8 μmol/L) alone had minimal impact on PC-9/ER and H1975 cell proliferation. However, co-treatment with SAHA and erlotinib significantly reduced viability in both cell lines. Combination index (CI) analysis confirmed synergy, with CI values <1 across most concentrations. For example, at 2 μmol/L SAHA + 2 μmol/L erlotinib, CI values were 0.65 and 0.58 for H1975 and PC-9/ER cells, respectively, indicating strong synergism.
PTEN Loss Correlates with Erlotinib Resistance
Western blot analysis revealed that PTEN protein expression was markedly reduced in PC-9/ER cells compared to parental PC-9 cells, whereas H1975 cells retained PTEN levels similar to PC-9. This suggests PTEN loss contributes specifically to acquired resistance rather than primary resistance mediated by EGFR mutations like T790M. To confirm SAHA’s role in modulating PTEN, PC-9/ER cells were treated with SAHA (0.25–4 μmol/L) for 48 hours. SAHA dose-dependently restored PTEN mRNA and protein levels, with 2 μmol/L SAHA increasing PTEN mRNA by 3.2-fold and protein by 2.8-fold compared to untreated controls.
Enhanced Apoptosis via SAHA and Erlotinib Combination
Flow cytometry using Annexin V-FITC/propidium iodide staining demonstrated that SAHA combined with erlotinib significantly increased apoptosis in resistant cells. In H1975 cells, the apoptosis rate rose from 10.12% ± 0.99% (erlotinib alone) and 29.8% ± 1.55% (SAHA alone) to 52.73% ± 2.63% with combination therapy. Similarly, in PC-9/ER cells, apoptosis increased from 16.09% ± 1.75% (erlotinib) and 14.25% ± 1.19% (SAHA) to 43.6% ± 4.99% with co-treatment. Western blotting corroborated these findings, showing elevated cleavage of caspase-3 and PARP, hallmark markers of apoptotic activation, in cells exposed to the drug combination.
Mechanistic Insights: PTEN Upregulation Restores Sensitivity
The study highlights PTEN’s pivotal role in modulating resistance. PTEN deletion in PC-9/ER cells likely drives resistance by hyperactivating the PI3K/Akt survival pathway, a known consequence of PTEN loss. SAHA’s ability to restore PTEN expression re-sensitizes cells to erlotinib by reinstating PTEN’s tumor-suppressive functions, including apoptosis induction and cell cycle regulation. Furthermore, SAHA’s HDAC inhibitory activity may epigenetically reactivate PTEN transcription, counteracting the silencing mechanisms that downregulate PTEN in resistant cells.
Clinical Implications and Future Directions
This work positions SAHA as a promising adjunct to EGFR-TKI therapy in NSCLC. By targeting epigenetic modifications and restoring PTEN expression, SAHA may delay or reverse acquired resistance, extending the therapeutic window for erlotinib. Future studies should validate these findings in vivo and explore whether PTEN status predicts response to HDAC inhibitor combinations. Additionally, investigating SAHA’s impact on other resistance mechanisms, such as MET amplification or epithelial-mesenchymal transition, could broaden its clinical applicability.
In conclusion, SAHA overcomes erlotinib-acquired resistance in NSCLC by restoring PTEN expression and enhancing apoptosis. This synergistic combination represents a viable strategy to improve outcomes in EGFR-TKI-resistant lung cancer, addressing a critical unmet need in oncology.
doi.org/10.1097/CM9.0000000000000823
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