Overcoming acquired resistance to cetuximab by dual targeting HER family receptors with antibody-based therapy

Abstract

Background: Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers. However, many patients who initially respond to cetuximab acquire resistance. To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (Ctx(R)) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in Ctx(R) clones due to decreased EGFR ubiquitination and degradation and 2) Ctx(R) clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the Ctx(R) setting.

Results: Since HER3 has been implicated in resistance to EGFR inhibitors, the efficacy of dually targeting both EGFR and HER3 in Ctx(R) models was evaluated. First, EGFR and HER3 expression were knocked down with siRNAs. Compared to the Ctx(S) parental cell line (HP), all Ctx(R) clones exhibited robust decreases in cell proliferation upon dual knockdown. Analysis of Ctx(R) clones indicated that neuregulin-1 was highly overexpressed compared to HP cells. Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab. Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects. Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT. Treatment with both mAbs resulted in knockdown of both signaling pathways simultaneously. HER2 was also strongly inactivated upon dual mAb therapy, suggesting that this treatment regimen can diminish signaling from three HER family receptors. De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo. Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. Combinatorial-treated xenograft tumors expressed decreased Ki67 and increased cleaved caspase-3 levels compared to tumors treated with either monotherapy.

Conclusions: These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.

Figure 1

Cetuximab-resistant (Ctx ^R ) clones have increased HER family receptor activity. (A) Ctx^R cells have increased HER family activity as well as phosphorylation levels of downstream signaling molecules. Parental (HP) and Ctx^R clones (HC1, HC4, HC8) were harvested and protein lysates were fractionated on SDS-PAGE followed by Immunoblotting for the indicated proteins. α-Tubulin was used as a loading control. (B) Expression of EGFR and HER3 on the plasma membrane is increased in Ctx^R clones compared to cetuximab-sensitive (Ctx^S) cells (HP). The levels of EGFR and HER3 on the cell surface were measured by flow cytometry. (n = 3) (C) EGFR displayed increased association with HER3 in all three Ctx^R clones compared to Ctx^S HP cell line. Cells were harvested and EGFR or HER3 were immunoprecipitated with anti-rabbit EGFR antibody or anti-rabbit HER3 antibody. The immunoprecipitate complexes were fractionated on SDS-PAGE followed by immunoblotting for indicated proteins.

Figure 2

Ctx ^R clones depend on EGFR and HER3 for proliferation and cetuximab response. (A) Effects of EGFR and HER3 knockdown on proliferation of Ctx^R cells. Proliferation was measured at 72 h after treatment using the crystal violet assay. Data points are represented as mean ± s.e.m (n = 3). *p ≤0.05. Whole cell lysates were harvested after 72 h treatment and fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control. (B) Upregulation of NRG-1 ligand in Ctx^R clones by real-time qPCR. NRG-1 expression level in Ctx^R clones HC1, HC4 and HC8 was measured by real-time qPCR analysis. Data are represented as fold increase relative to the HP parental control. Data points are represented as mean ± s.e.m. (n = 4). (C) NRG-1 ligands can enhance cetuximab resistance in cetuximab-sensitive cells. Proliferation was measured at 72 h after treatment using CCK8 assays and plotted as a percentage of proliferation relative to the vehicle cells. Data points are represented as mean ± s.e.m. (n = 4). (D) NRG-1 increased phosphorylation levels of HER family receptors and their respective kinase targets in HP cells. Whole cell lysates were harvested and fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control.

Figure 3

U3-1287 downregulates total and phosphorylation of HER3 as well as AKT phosphorylation, but does not inhibit cell proliferation in Ctx ^R clones. (A) U3-1287 alone did not inhibit cell proliferation in Ctx^R clones. The cell proliferation was measured via crystal violet assay and plotted as a percentage of proliferation relative to the vehicle control cells. Data points are represented as mean ± s.e.m. (n = 3). (B) U3-1287 downregulates total HER3 and phosphorylation of AKT in Ctx^R clones. Whole cell lysates were fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control.

Figure 4

Dual blockade of HER3 and EGFR can effectively inhibit the proliferation of Ctx ^R clones. (A) Combinatorial treatment of Ctx^R clones with cetuximab and U3-1287 leads to proliferation inhibition. Cell proliferation was measured using crystal violet assay and plotted as a percentage of proliferation relative to the vehicle control cells. Data points are represented as mean ± s.e.m. (n = 3). (B) Combinatorial treatment with cetuximab and U3-1287 leads to loss of HER3 expression in Ctx^R clones. Protein lysates were fractionated on SDS–PAGE followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control.

Figure 5

Combined treatment of Ctx ^R clones with cetuximab and U3-1287 inhibits HER2, AKT and MAPK signalings more effectively than either drug alone. (A) Human Phospho-Kinase array analysis demonstrated that combined treatment with cetuximab and U3-1287 inhibits proliferation and survival signaling in Ctx^R cell clone, HC4. The cell extracts were incubated with membranes containing antibodies to 46 different kinase phosphorylation sites. Quantitation of phosphorylated proteins was completed using scanned images from ImageJ software. Data points are represented as the mean of duplicate spots. (B) Effects of combined cetuximab and U3-1287 treatment on their respective kinase targets in Ctx^R clones. Protein lysates from Figure 5A (HC4) were fractionated on SDS–PAGE followed by immunoblotting for the indicated proteins. Protein lysate from other Ctx^R clones (HC1 and HC8) as well as HP cells were obtained after treatment with vehicle, cetuximab (20 ug/mL), U3-1287 (100 ug/mL) or the combination of cetuximab and U3-1287 for 24 h. α-Tubulin was used as a loading control.

Figure 6

Cetuximab and U3-1287 induced apoptosis in Ctx ^R clones. (A) Combinatorial treatment with cetuximab and U3-1287 activates Caspase 3/7 compared to either drug alone in Ctx^R clones. Caspase-3/7 activity was determined by Caspase 3/7-Glo assay. Data represent means ± s.e.m from 3 independent experiments (n = 9). *p <0.05 or **p <0.01. (B) The percentage of Annexin v-positive/propidium iodide-negative cells was increased significantly after the combination of cetuximab and U3-1287 treatment compared to vehicle, cetuximab or U3-1287 alone in the Ctx^R clones. Cells were plated and allowed to adhere for 24 h prior to treatment with vehicle, cetuximab (20 ug/mL), U3-1287 (50 ug/mL) or the combination of cetuximab (20 ug/mL) and U3-1287 (50 ug/mL) for 24 h prior to Annexin-v analysis via flow cytometry. Data points are represented as mean ± s.e.m. (n = 3). *p <0.05 or **p ≤0.001. Flow cytometry profile in HC4 cells represents Annexin-V-FITC staining in x-axis and PI in y-axis. The number represents the percentage of cells in each condition.

Figure 7

Combination of cetuximab and U3-1287 treatment of Ctx ^R tumors leads to growth delay in vivo. (A) Growth-delay effects of U3-1287 in Ctx^R tumors in vivo. The black arrow designates the starting time point of U3-1287 treatment. The average tumor volume of mice treated with IgG is included in all groups for comparison purposes. (B) Combination treatment with cetuximab and U3-1287 inhibited HER3 expression and HER2 activation in vivo. Total and phosphorylation levels of HER2 and HER3 proteins in Ctx^R xenograft tumors were determined by immunoblot analysis after cetuximab, U3-1287 or the combination of cetuximab and U3-1287 treatments. (C) The inhibition of phospho-HER3 and phospho-HER2 expression in Ctx^R tumors after combinatorial treatment corresponds with reduced proliferation and increased apoptosis. Ctx^R tumor samples after cetuximab, U3-1287 or the combination of cetuximab and U3-1287 treatment in vivo were prepared and analyzed for Ki67 and cleaved caspase 3 by immunohistochemistry. Images were quantified via taking the average staining intensity measured from 3 tumors per treatment group (3 images/tumor, n = 9). Magnification 100X.