Mapping C-terminal transactivation domains of the nuclear HER family receptor tyrosine kinase HER3.

Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B₁) and 27 (B₂) amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B₁ and B₂ regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B₁ and B₂ regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs

Nuclear HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain.

<p><b>A. HER3 knockdown decreases the activation of the cyclin D1 promoter.</b> H226^R, SKBr3, and MCF-7 cells were incubated with non-targeting (NT) or HER3 siRNA for 24 hr followed by transfection with the 122 bp cyclin D1 promoter-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 6). Percent decreases in cyclin D1 promoter-luciferase activity were normalized to NT transfected cells. The graph is representative of three independent experiments. Luciferase lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. <b>B. HER3 overexpression activates the cyclin D1 promoter.</b> SCC6, HCC1954, SKBr3, and BT474 cells were transfected with HER3WT or control vector, 122 bp cyclin-D1-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1-promoter luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Nuclear lysate was harvested from each cell line and fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for the Nuc fraction. <b>C. HER3ΔB₁ΔB₂ overexpression can prevent the activation of the cyclin D1 promoter while HER3DM remains functional.</b> SCC6, BT474 and HCC1954 cells were transfected with HER3WT, HER3DM, HER3ΔB₁ΔB₂ or control vector, the 122 bp cyclin-D1-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of seven independent experiments. <b>Inset 1:</b> CHOK1 cells were transfected with HER3WT, HER3ΔB₁ΔB₂ or control vector for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. <b>Inset 2:</b> CHOK1 cells were transfected with HER3WT, HER3DM, HER3ΔB₁ΔB₂ or control vector and stimulated for 40 min with 5 nM neuregulin-1. Whole cell lysate was fractionated on SDS-PAGE followed by immunoblotting for indicated proteins. <b>D. HER3 knockdown decreases cyclin D1 expression.</b> SCC6 and BT474 cells were incubated with NT or HER3 siRNA for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to NT transfected cells. The graph is representative of two independent experiments. <b>E. The overexpression of HER3WT enhances cyclin D1 expression while HER3ΔB₁ΔB₂ is hindered.</b> SCC6 and BT474 cells were transfected with HER3WT, HER3ΔB₁ΔB₂ or control vector for 72 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of four independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of <i>Renilla</i> luciferase. All data points are represented as mean +/− s.e.m. P<0.05.</p

Confocal Immunofluorescent staining of nuclear HER3.

<p>C-TERM and N-TERM HER3 antibodies were used to visualize HER3 localization in H226^R, SKBr3, and BT549 cells. Alexa Fluor-546 secondary antibody was used to visualize HER3 (RED) and DAPI was used to visualize the nucleus (BLUE). Merged images were magnified to depict the nuclear localization of HER3 (see white arrows). Magnification 600X.</p

HER3 can associate with the cyclin D1 promoter.

<p><b>A. Nuclear EGFR and HER2 associate with the cyclin D1 promoter.</b> ChIP using an anti-EGFR, anti-HER2, or normal rabbit IgG antibody was performed with SKBr3 cells and isolated DNA was subsequently used for qPCR with primers flanking the 122 bp cyclin D1 promoter region (n = 3). qPCR specificity for the 122 bp cyclin D1 promoter was confirmed by agarose gel electrophoresis of semi-qPCR products. DAPA analysis was performed using a biotinylated 122 bp cyclin D1 promoter probe incubated with 400 ug of nuclear lysate harvested from SKBr3 cells. Bound proteins were isolated with streptavidin agarose beads and subsequently fractionated on SDS-PAGE followed by immunoblotting for EGFR or HER2. Nuclear lysate incubated with beads only lacked association. <b>B. Nuclear HER3 can associate with the cyclin D1 promoter.</b> ChIP using a N-TERM anti-HER3 or a human IgG antibody was performed with H226^R, SKBr3, and BT549 cells. qPCR was performed as in 5A. <b>C. HER3 can associate with a cyclin D1 promoter probe.</b> DAPA analysis was performed using nuclear lysate harvested from H226^R, SKBr3, MCF-7, and BT549 cells as in 5A. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. <b>D. HER3 association with the cyclin D1 promoter probe is specific.</b> DAPA analysis was performed as in 5A using nuclear lysate harvested from H226^R, SKBr3, MCF-7, and BT549 cells transfected with either non-targeting (NT) or HER3 siRNA for 48 hr. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. All data points for ChIP are represented as mean +/− s.e.m and normalized to the IgG control. P<0.05.</p

The C-terminus of HER3 contains a strong transactivation domain.

<p><b>A. EGFR intracellular domain (ICD) map and plasmid validation.</b> The intracellular domain (ICD) and C-terminal domain (CTD) of EGFR were fused to the Gal4 DNA binding domain (Gal4DBD). CHOK1 cells were transfected with each construct for 48 hr prior to harvesting WC lysate and fractionation on SDS-PAGE followed by immunoblotting for EGFR and Gal4DBD. EGFRWT vector was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control<b>. B. EGFR-CTD contains strong transactivation potential.</b> CHOK1 cells were transfected with EGFR-ICD or EGFR-CTD constructs, UAS-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. <b>C. HER3-ICD map and plasmid validation.</b> The HER3- ICD, juxtamembrane and tyrosine kinase domain (JKD), and CTD were fused to the Gal4DBD. Transfection was performed the same as in 1A and immunoblot analysis was performed for HER3 and Gal4DBD. HER3WT was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control. <b>D. HER3-CTD contains strong transactivation potential.</b> CHOK1, H226^R, and SKBr3 cells were transfected with HER3-ICD, HER3-JKD, and HER3-CTD constructs, UAS-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. Luciferase activity was normalized to DNA content, protein content, and the expression of <i>Renilla</i> luciferase in all assays. Luciferase activity detected for each construct was normalized to the Gal4DBD vector control. Data points are represented as mean+/−s.e.m. p<0.05. TM (transmembrane); JM (juxtamembrane); KD (kinase domain).</p

The HER3 receptor is localized to the nucleus in its full-length form.

<p><b>A. HER3 is expressed in numerous cancer cell lines.</b> Whole cell protein lysates were isolated from various breast, lung, HNSCC, and colon cancer cell lines. Lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin was used as a loading control. <b>B. HER3 is localized to the nucleus in cancer cell lines.</b> H226^R, SKBr3, MCF-7, and BT549 cells were harvested for whole cell (WC), non-nuclear (NN) and nuclear (Nuc) protein, fractionated on SDS-PAGE followed by immunoblotting for HER3. Clathrin, dynamin, calnexin, α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. <b>C. Specificity of nuclear HER3 by siRNA.</b> H226^R and SKBr3 cells were harvested for NN and Nuc protein 48 hr post treatment with siHER3 or non-targeting (NT) siRNA. Experimental procedure as in 1B. α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. <b>D. Full-length HER3 is localized to the nucleus.</b> H226^R and SKBr3 cells were harvested for WC, NN, and Nuc lysate. WC lysates were harvested 48 hr post treatment with siHER3. 250 ug of cell lysate was immunoprecipitated with an N-TERM HER3 antibody or human IgG control. The immunoprecipitates were fractionated on SDS-PAGE followed by immunoblotting for HER3 with a C-TERM antibody.</p

An intracellular domain (ICD) mutant of HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain.

<p><b>A. HER3WT ICD (WT-ICD) can activate the cyclin D1 promoter while HER3ΔB₁ΔB₂-ICD (ΔB₁ΔB₂-ICD) is hindered.</b> SCC6, BT474, and HCC1954 cells were transfected with WT-ICD, ΔB₁ΔB₂-ICD or control vector, the 122 bp cyclin D1-luciferase and Tk-<i>Renilla</i> reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. <b>Inset 1:</b> Illustration of both the WT-ICD and ΔB₁ΔB₂-ICD constructs. <b>Inset 2:</b> CHOK1 cells were transfected with WT-ICD and ΔB₁ΔB₂-ICD for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. <b>B. The overexpression of WT-ICD can enhance cyclin D1 expression while ΔB₁ΔB₂-ICD is hindered.</b> SCC6 and BT474 cells were transfected with WT-ICD, ΔB₁ΔB₂-ICD or control vector for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of three independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of <i>Renilla</i> luciferase. All data points are represented as mean +/− s.e.m. P<0.05. ICD (intracellular domain).</p

Sym004, a Novel EGFR Antibody Mixture, Can Overcome Acquired Resistance to Cetuximab

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don't respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resistance using the non-small cell lung cancer line NCI-H226. These cetuximab-resistant (CtxR) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of antibodies directed against distinct epitopes on the extracellular domain of EGFR, as an alternative therapy for CtxR tumor cells. Sym004 treatment of CtxR clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo CtxR NCI-H226 mouse xenografts and subsequently treated CtxR tumors with Sym004. Sym004 treatment of mice harboring CtxR tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly decreased in CtxR tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for CtxR tumors