The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

Abstract

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

Fig 1. Synergistic growth inhibitory effects of panobinostat (LBH589) combined with lapatinib (LAP) in CRC cell lines

Growth inhibition was determined by MTS assay for 72 h. Six CRC cell lines were exposed to increasing doses of LBH589 and LAP. Data points represent mean±SD percent growth inhibition (n=3 experiments) compared to controls at 100%. The combined drug effect was analyzed using the combination index (CI) equation and presented with fraction affected (FA) for combinations. CI values <1=synergism; 1–1.2=additive; and >1.2=antagonism.

Fig 2. Panobinosat (LBH589) and lapatinib (LAP) synergistically suppress colony formation and induce apoptosis in CRC cell lines

(A) DLD-1, H630, HCT116 and LoVo CRC cells were treated with LBH589 and 3μM LAP for 24h and then replaced with drug-free media for 12–15 days. Data presented as the percentage of colony formation compared to controls, mean±SEM from (n=3 experiments). Statistical significance was determined by two-way ANOVA, *p<0.05;**p<0.01,***p<0.001. (B) Percentage of cells in sub-G₁ at 24h are represented as the mean±SD from two independent experiments, *p<0.05;**p<0.01;***p<0.001 two-way ANOVA. (C) Western blot analysis of γ-H2A.X, caspase-8 and PARP following treatment for 18 and 24h. β-Tubulin controlled for loading.

Fig 3. Panobinostat (LBH589) and lapatinib (LAP) synergistically enhance antitumor activity in LoVo CRC xenograft model

Male nu/nu mice with subcutaneous LoVo CRC tumors (100 mm³) were treated with vehicle, LAP, LBH589 or their combination (n=6, group). 30 mg/kg LAP was administered by oral galvage twice daily and 2.5 mg/kg LBH589 was administered i.p. for 5 consecutive days per week. (A) Tumor volume (TV) is represented as mean±SEM for each group. Statistical significance was determined by two-way ANOVA, *p<0.05;**p<0.0;***p<0.001. (B) Tumor delay (Td)¹=time in days to reach a tumor volume 5 times the initial volume on day 1. Expected Td²=the mean vehicle + (mean LAP) - mean vehicle) + (mean LBH589) - mean vehicle). Observed Td³=the combination of 30 mg/kg LAP and 2.5 mg/kg LBH589. Ratio⁴ of observed:expected Td. Ratio >1 indicates synergism and <1 indicates antagonism. (C) Mouse bodyweight represented as the percent initial body weight at day 24 compared to day 1.

Fig 4. Panobinostat (LBH589) modulates EGFR and HER2 mRNA and protein expression

DLD-1, H630, HCT116, and LoVo CRC cell lines were treated with LBH589 for 24h. Following treatment mRNA expression was determined for (A) ERBB1 and (B) ERBB2. Histogram bars represent the mean±SD for (n=2 analyzed in triplicate). GAPDH mRNA expression was used to normalize. Statistical significance was determined by student t-test of treated samples when compared to control, *p<0.05;**p<0.01;***p<0.001. (C) Western blot analysis of the effect of LBH589 on EGFR and HER2 protein expression. Densitometric analysis was performed using Scion Image normalized to β-tubulin to control for loading.

Fig 5. Significant suppression of HER family and downstream signaling molecules following combination treatment with panobinostat (LBH589) and lapatinib (LAP)

H630 and LoVo cells were treated with LBH589 and LAP for 18 and 24h and analyzed for mRNA and protein expression of HER family and downstream signaling. mRNA expression was determined for (A) ERBB1, ERBB2, CCND1, NFκB1, and IRAK1. Histogram bars represent the mean±SD (n=2 analyzed in triplicate). GAPDH expression was used to normalize mRNA expression. Statistical significance was determined by student t-test of treated samples when compared to controls, *p<0.05;**p<0.01;***p<0.001. (B) H630 and (C) LoVo cell lines were evaluated for the effects of treatment on HER pathway proteins by Western blot analysis. β-Tubulin controlled for loading.

Fig 6. Combined panobinostat (LBH589) and lapatinib (LAP) treatment downregulates HER3 mRNA and protein in CRC cell lines

(A) Basal HER3 mRNA and protein levels were determined in six CRC cell lines. Histogram represents mean±SD (n=2 analyzed in triplicate). Protein expression was determined by Western blotting. Densitometeric analysis was performed using Scion Image and normalized to β-tubulin to control for loading. Relative protein expression is normalized to HCT116. (B) H630 and LoVo cells were treated with LBH589 for 24h and HER3 mRNA and protein analyzed. Statistical significance was determined by student t-test of treated samples when compared to respective control control, *p<0.05;**p<0.01;***p<0.001. (C) The effects of the LAP plus LBH589 at 24h on HER3 mRNA and protein expression was analyzed as above.