Radiosensitization and downregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon inhibition of mitogen/extracellular signal-regulated kinase (MEK) in malignant melanoma cells

Abstract

Background: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an important cofactor in the p53-mediated DNA damage response pathway upon ionizing radiation (IR) and exerts anti-apoptotic effects also independent of p53 pathway activation. Furthermore, hnRNP K is overexpressed in various neoplasms including malignant melanoma (MM). Here, we investigate the role of hnRNP K in the radioresistance of MM cells.

Methods and results: Our results show cytoplasmic expression of hnRNP K in human MM surgical specimens, but not in benign nevi, and a quick dose- and time-dependent upregulation in response to IR accompanied by cytoplasmic redistribution of the protein in the IPC-298 cellular tumor model carrying an activating NRAS mutation (p.Q61L). SiRNA-based knockdown of hnRNP K induced a delayed decline in γH2AX/53BP1-positive DNA repair foci upon IR. Pharmacological interference with MAPK signaling abrogated ERK phosphorylation, diminished cellular hnRNP K levels, impaired γH2AX/53BP1-foci repair and proliferative capability and increased apoptosis comparable to the observed hnRNP K knockdown phenotype in IPC-298 cells.

Conclusions: Our results indicate that pharmacological interference with MAPK signaling increases vulnerability of NRAS-mutant malignant melanoma cells to ionizing radiation along with downregulation of endogenous hnRNP K and point towards a possible use for combined MEK inhibition and localized radiation therapy of MM in the NRAS-mutant setting where BRAF inhibitors offer no clinical benefit.

Keywords: MEK inhibition; NRAS; melanoma; nRNP K; radiotherapy.

Figure 1

A. Expression of hnRNP K in malignant melanoma tissue specimens. Criteria for classifying nuclei (N) and cytoplasm (C) as positive or negative for hnRNP K are illustrated by representative immunohistochemistry for hnRNP K. B. IHC scoring was done by separately analyzing hnRNP K staining intensity for nuclear and cytoplasmic hnRNP K expression. Mean nuclear IHC scores of benign nevi, malignant melanoma (MM) or MM metastasis showed no significant difference. 25% of MM and MM metastases stained positively for cytoplasmic hnRNP K whereas nevi were completely devoid of cytoplasmic hnRNP K expression (n: nevi = 18, melanoma = 62, metastasis = 20; *p < 0.05 versus nevi). C. Verification of NRAS mutation in IPC-298 cells by DNA pyrosequencing. The CAA to CTA transition leads to a p.Q61L amino acid substitution, comprising constitutive signaling activity along the MAPK pathway. D. Inhibition of MEK signaling by 50 μM PD98059 abrogates phosphorylation of ERK1/2 and reduces endogenous hnRNP K levels.

Figure 2

A. Immunoblotting of hnRNP K shows dose-dependent upregulation within 30 minutes after IR. B. Immunoblotting of IPC-298 cell lysate showing time-dependent increase in intensity of the hnRNP K-double band with a maximum at 30 – 60 minutes and a subsequent normalization after 180 minutes, parallel to the detection of phospho-ATM. C. 6 hours and 24 hours after applying IR (2 Gy), there is no difference in hnRNP K expression between irradiated and non-irradiated samples. D. Immunofluorescence microscopy of IPC-298 cells shows strict nuclear localization of hnRNP K under control conditions. Within 30 minutes, exposure to IR causes perinuclear accumulation of hnRNP K in a dose-dependent manner. Scale bars represent 30μm. E. DNA damage repair as illustrated by dose-dependent increase in γH2AX/53BP1-Foci (n = 100; p < 0.05). F. Subcellular fractionation and immunoblotting reveals cytoplasmic (Cyt) accumulation of hnRNP K 30 min post-IR (2 Gy). Nuclear (Nuc) hnRNP K levels remain unchanged.

Figure 3

A. Immunoblotting shows effective knockdown of hnRNP K. B. Knockdown of hnRNP K and MEK inhibition result in a significantly reduced decline in γH2AX/53BP1 foci (*p < 0.05). C. Time-dependent DNA-damage response (DDR) shown by γH2AX/53BP1 foci upon 2 Gy IR. Co-localization of γH2AX/53BP1 was verified by staining intensity profile. D. Knockdown of hnRNP K evokes radiosensitization of IPC-298 cells (n = 4). F. MEK inhibition with PD98059 impairs clonogenic viability of IPC-298 melanoma cells in a dose-dependent manner (n = 4). G. Representative colony formation assays show partial depletion of clonogenic cell survival by treatment with 5 μM PD98059 and complete abrogation by additional irradiation with 2 Gy (n = 4).

Figure 4

A and B. MEK inhibition with PD98059 for 24 hours post-IR impairs normalization of cellular hnRNP K levels in irradiated and non-irradiated IPC-298 cells comparable to the hnRNP K knockdown phenotype. C. Effects of MEK inhibition and hnRNP K knockdown on apoptosis were analysed by flow cytometry using Annexin V-FITC/PI staining. Presented data show the results of a contiguous test series. Early apoptosis is represented by Annexin V⁺/PI⁻ events, whereas Annexin V⁺/PI⁺ counts demonstrate late apoptosis/necrosis. D. Statistical analyses of flow cytometry results. Experiments were performed in triplicate. Untreated cells served as control. The presented columns are given as the means ± S.D. (*p < 0.05, n.s. = not significant, ^*p = 0.05).

Figure 5

Schematic model for the role of MAPK-mediated upregulation of hnRNP K in the DDR pathway and possible targets for pharmacological interference.