Abstract 4456: Increased NTF2 levels in melanoma cell lines affect nuclear size and cancer cell characteristics
Authors: Vukovic Lidija D. , Stohr Bradley A. , Levy Dan L. 
Published: August 2018

Xenopus studies have implicated two nuclear transport factors in the regulation of nuclear size: NTF2 and importin α. Importin α, a positive regulator of nuclear size, has been suggested as a biomarker in breast cancer and non-small cell lung carcinoma. We have focused our studies on NTF2. In different staged melanoma cell lines and tissue samples, we observed an inverse correlation between NTF2 levels and nuclear size. Compared to cancer cells, normal melanocytes exhibited the smallest nuclei and highest NTF2 levels. Based on transient transfection experiments in HeLa cells, MRC5 cells, and different melanoma cell lines, we found that increasing NTF2 expression levels reduces nuclear size. The largest effect was observed in WM3211 melanoma cells in which NTF2 expression reduced nuclear cross-sectional area by 40%. We next generated a stably transfected NTF2-inducible version of the metastatic melanoma cell line WM983B. By altering the doxycycline concentration, we can titrate the level of NTF2 protein, and we found that 20 ng/ml of doxycycline increases NTF2 levels by 5-fold and decreases the nuclear cross-sectional area by about 10%. Doxycycline-treated cells exhibited 3-fold higher rates of apoptosis and a 40% reduction in the rate of cell migration in a wound-healing assay. In vivo experiments were performed on RAG2 and NSG mice where NTF2-inducible cells were injected intravenously in order to examine metastatic potential. The number of lung metastases was reduced by 7-fold in doxycycline-treated mice compared to those that were not treated, suggesting that NTF2 overexpression suppresses metastatic potential. We are now testing the hypothesis that altering nuclear size by NTF2 expression leads to chromatin remodeling and altered gene expression. We performed RNAseq on doxycycline-treated and non-treated cells. Differentially expressed genes include those involved in cell migration (e.g. PRKX, LAMA5, ADAMTS15, ANK3, CERS6) and cell survival (e.g. RAMP1, WNK3, BMP5, TP63). Performing DNA FISH will show if the nuclear positioning of these genes is changed when nuclear size is reduced. We are now planning to test a combination therapy in mice in which we reduce nuclear size and treat with the well-established melanoma drug vemurafenib. Most melanoma patients with BRAF mutations show a good response to this drug, however relapse often occurs with drug-resistant melanoma cells expressing high levels of the cancer stem cell markers JARID1B, CD271, and fibronectin. According to our RNAseq data, cells expressing higher levels of NTF2 show reduced gene expression of JARID1B and fibronectin. Thus increasing NTF2 expression in drug-resistant tumors may improve prognosis and survival time.

Citation Format: Lidija D. Vukovic, Bradley A. Stohr, Dan L. Levy. Increased NTF2 levels in melanoma cell lines affect nuclear size and cancer cell characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4456.