Comparative analysis of cancer cell responses to targeted radionuclide therapy (TRT) and external beam radiotherapy (EBRT)

Abstract

The vast majority of our knowledge regarding cancer radiobiology and the activation of radioresistance mechanisms emerged from studies using external beam radiation therapy (EBRT). Yet, less is known about the cancer response to internal targeted radionuclide therapy (TRT). Our comparative phosphoproteomics analyzed cellular responses to TRT with lutetium-177-labeled minigastrin analogue [¹⁷⁷Lu]Lu-PP-F11N (β-emitter) and EBRT (ɣ-rays) in CCKBR-positive cancer cells. Activation of DNA damage response by p53 was induced by both types of radiotherapy, whereas TRT robustly increased activation of signaling pathways including epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs) or integrin receptor. Inhibition of EGFR or integrin signaling sensitized cancer cells to radiolabeled minigastrin. In vivo, EGFR inhibitor erlotinib increased therapeutic response to [¹⁷⁷Lu]Lu-PP-F11N and median survival of A431/CCKBR-tumor bearing nude mice. In summary, our study explores a complex scenario of cancer responses to different types of irradiation and pinpoints the radiosensitizing strategy, based on the targeting survival pathways, which are activated by TRT.

Keywords: CCKBR; Erlotinib; Minigastrin; Phosphoproteomics; Radioresistance.

Fig. 1

Cellular responses to TRT and EBRT. a A431/CCKBR cells were treated with [¹⁷⁷Lu]Lu-PP-F11N or exposed to EBRT and the generated tryptic peptides and phosphopeptide enriched samples were subjected to mass spectrometry analysis. b Charts display phosphopeptide (upper panels) and protein (lower panels) abundance changes shown as log2 transformed fold change (FC). Red dots represent phosphopeptides or proteins with significantly altered abundance. FDR < 0.05. c Hierarchical clustering of identified changes in phosphopeptide and protein abundance. d Number of phosphopeptides and protein abundance changes. Arrows indicate up-regulated (purple) or down-regulated (blue) phosphorylation or protein levels. e MS2-based quantification of abundance changes was shown as Log2 transformed radiation/control ratios for CYR61, GAPDH, ERK2 and P53 protein levels and phosphorylation of ERK, P53 and JUN after exposure to [¹⁷⁷Lu]Lu-PP-F11N and EBRT in A431/CCKBR cells. f, g Expression and phosphorylation levels of the same proteins were validated by quantitative WB analysis on total protein lysates isolated from untreated (control) and [¹⁷⁷Lu]Lu-PP-F11N- or EBRT-treated cells. Each treatment was performed in triplicate (Exp 1–3). Quantification of signal intensities (in f) is shown as Log2 transformed radiation/control ratios as described above. *P < 0.05, **P < 0.01, ***P < 0.001. h WB analysis for CYR61 and P53 phosphorylation in total protein lysates isolated from A431/CCKBR cells untreated (control) and treated with 1, 2, 4, 6 and 8 Gy at indicated time points. Blot was re-probed with antibody against GAPDH and total P53

Fig. 2

Validation of radiosensitizing targets to TRT. a Reduced proliferation of A431/CCKBR cells treated with kinase inhibitor library in combination with [¹⁷⁷Lu]Lu-PP-F11N, shown as log2 [ratio: combinatory treatment/[¹⁷⁷Lu]Lu-PP-F11N monotherapy]. Red dots represent inhibitors, which significantly increased therapeutic response to [¹¹⁷Lu]Lu-PP-F11N (P < 0.05). b Cell proliferation 48 h after treatment with 10 µM STS, BML-265 and TYRPHOSTIN AG 1478 alone or in combination with 5 MBq per ml of [¹⁷⁷Lu]Lu-PP-F11N. c A431/CCKBR cell proliferation was analyzed 72 h after treatment with 20 µM cilengitide (CGT) and/or 5 and 10 MBq per ml of [¹⁷⁷Lu]Lu-PP-F11N, as indicated. Results were assayed in triplicate and proliferation in control untreated cells was set to 1. Data represent means ± SD. d Experimental design of in vivo study: After implantation of A431/CCKBR cells into nude mice, 10 doses of cilengitide (CGT) or erlotinib were administrated alone or in combination with 60 kBq [¹⁷⁷Lu]Lu-PP-F11N, as indicated. e Tumor growth curves of control and treated groups. Data represent mean ± SD. f Survival rates presented as Kaplan–Meier curves of control and treated mice. g Extended median survival in treated groups as compared to control mice. *P < 0.05, **P < 0.01, ***P < 0.001