Photo-activated psoralen binds the ErbB2 catalytic kinase domain, blocking ErbB2 signaling and triggering tumor cell apoptosis

Abstract

Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85(ErbB2)) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85(ErbB2). Here we show that PUVA reduced p85(ErbB2) phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

Figure 1. PUVA antitumor activity in HER2 + breast cancer cells.

Cells were pre-treated with the indicated concentrations of 8MOP for 4 hr before UVA irradiation (2J), and then cultured for an additional 72 hr before being analyzed for cell growth (A) BT474; (B) SKBR3; (C) MCF7, (D) HFF and apoptosis (D) BT474; (E) SKBR3. Cells treated with vehicle (0.01% DMSO) alone served as controls. Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments.

Figure 2. PUVA therapy inhibits ErbB2 signaling.

BT474, SKBR3 and MCF7 cells were subjected to the indicated treatment conditions as described in Figure 1. Western blot analysis was performed on whole cell lysates. Actin steady-state protein levels served as a control to ensure for equal loading of protein. Results are representative of three independent experiments.

Figure 3. Inhibition of ErbB2 signaling in response to PUVA is independent of DNA crosslinking.

The chemical structures of (A) 8MOP, and (B) 7-methylpyridopsoralen (SMSF032310), which is a derivative of 8MOP that lacks the ability to crosslink DNA, are shown. (C) BT474 and SKBR3 cells were exposed to the indicated treatments. Cell growth and viability assays were performed after 72 hr. P<0.0003 (SKBR3); P<0.0009 (BT474). Cells treated with vehicle alone served as controls. Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. Corresponding Western blot analysis of the indicated protein/phosphoproteins is shown. Steady-state actin protein levels served as controls for equal loading of proteins. Results are representative of three independent experiments.

Figure 4. 8MOP interacts with the catalytic kinase domain of ErbB2.

(A) 8MOP interacts with three peptide regions within the ErbB2 catalytic kinase domain. Qualitative peptide identifications within the ErbB2 catalytic kinase domain following LC-MS/MS analysis of a streptavidin pull-down of biotinylated-8MOP bait (see Materials and Methods). The transmembrane domain is indicated (red diamond) and the five C-terminus tyrosine autophosphorylation sites are indicated (p). (B) Non-reducing Western blot analysis of the interaction of 8MOP with ErbB2. BT474 cells were treated with 800dye-8MOP (Promega) or with vehicle (0.01% DMSO) alone served as control for 48 hr and then exposed to UV irradiation (2J) prior to Western blot analysis. The image on the left shows the Western blot for ErbB2 (red). The image on the right shows the same membrane directly scanned for the presence of 800dye-8MOP (green), which overlays the ErbB2 signal. The results are representative of three independent experiments.

Figure 5. The combination of PUVA with the irreversible pan-ErbB TKI neratinib results in enhanced antitumor activity.

The growth and viability of BT474 and SKBR3 cells (top bar graphs) after being subjected to the indicated treatment conditions. The combination of PUVA plus neratinib: P<0.0005 (BT474 and SKBR3 cells). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. (B) Western blot analysis showing steady-state ErbB2, ErbB3, and phospho-Akt (S473) protein levels in BT474 and SKBR3 cells treated according to the indicated treatment conditions. Vehicle alone (0.01% DMSO) served as a control. Steady-state actin protein levels served as a control for equal loading of protein. The results are representative of three independent experiments.

Figure 6. PUVA therapy reverses acquired resistant to ErbB2 targeted therapies.

(A) Equal numbers of rBT474 and rSKBR3 cells were subjected to the indicated treatment conditions, and the effects on cell growth and viability are shown. P values of statistical significance are indicated. Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. (B) The corresponding Western blot analysis for each of the indicated treatment conditions is shown. As indicated, rBT474 and rSKBR3 cells are continuously maintained in 1 µM lapatinib. Actin steady-state protein levels served as a control to ensure for equal loading of protein. Results are representative of three independent experiments.

Figure 7. PUVA therapy targets nuclear p85^ErbB2, inducing tumor cell apoptosis.

Top bar graph shows the results of the growth assays performed in T47D and stably transfected T47D cell line. T47D cells expressing p85^ErbB2 were pretreated with 5 µM lapatinib or 5 µM 8MOP for 4 hr followed by irradiation in a UV Stratalinker 1800 (Statagene). Cells transfected with empty vector (T47D/Vector), and those treated with vehicle alone (0.01% DMSO) served as controls. The effects of the treatments on cell growth and viability are shown in the bar graph. P<0.0071 (8MOP + UVA irradiation). Results represent the mean +/− standard error of triplicate samples, and are representative of three independent experiments. Steady-state phospho-p85^ErbB2 protein levels (dotted arrow) and phospho-p185^ErbB2 (solid arrow) are shown by Western blot. Actin steady-state protein levels served as a control to ensure for equal loading of protein. Results are representative of three independent experiments.