Abstract 2433: Altering nuclear size impacts cancer cell characteristics in melanoma cell lines
Authors: Lidija D. Vukovic, Bradley A. Stohr, Dan L. Levy
Journal: Cancer Research (2017)

Abstract Nuclear size is altered in cancer cells, a change used by pathologists to distinguish cancer from normal cells. Previous studies in Xenopus identified two nuclear transport proteins, importin α and NTF2, as regulators of nuclear size. In general, importin α and NTF2 levels positively and negatively affect nuclear size, respectively. Increased importin α expression is used as a biomarker in non-small cell lung carcinoma and breast cancer and correlates with increased nuclear size. We identified decreased NTF2 protein expression as a potential biomarker in melanoma, consistent with NTF2 levels negatively regulating nuclear size. Following up on this result, our current studies focus on manipulating nuclear size in melanoma cells to assess the impact on cancer cell characteristics, including proliferation rate, migration potential, and apoptosis. First, we measured nuclear size and NTF2 expression levels in different stage melanoma cell lines. Compared to a normal melanocyte cell line, we consistently observed larger nuclei and lower NTF2 protein levels in all melanoma cell lines examined. Next, we performed transient transfections in HeLa, MRC5, and melanoma cell lines, demonstrating that ectopic NTF2 expression leads to reduced nuclear size. The most dramatic effects were observed in primary melanoma cell line WM3211 where higher NTF2 expression caused a 40% reduction in nuclear cross-sectional area. To obtain more precise control over NTF2 expression levels, we generated a stably transfected metastatic melanoma cell line (WM983B) in which NTF2 expression can be titrated by varying the concentration of doxycycline in the growth media. Higher doxycycline levels lead to increased NTF2 expression and smaller nuclei. In particular, 20 ng/ml doxycycline induced a 10% reduction in nuclear cross-sectional area. In a wound healing assay, doxycycline-treated cells exhibited a 40% reduction in cell motility compared to non-treated cells or the parent cell line treated with doxycycline. Furthermore, the doxycycline-treated cells exhibited an increased rate of apoptosis. We propose that reduced nuclear size in the doxycycline-treated cells causes changes in chromatin organization and gene expression, thus giving rise to observed effects on cell migration and apoptosis. To test this idea, we are currently performing a global transcriptomics analysis of our cell lines to identify genes whose expression is altered by nuclear size. Using this information, we will use DNA FISH to map the intranuclear position of differentially-expressed genes as a function of nuclear size. Also in vivo experiments are ongoing in which we have subcutaneously injected NTF2-inducible melanoma cells into NSG mice in order to examine tumor formation and metastatic capacity of cells with differently-sized nuclei. Regarding that in most cancer cells nuclear size is enlarged, understanding the causes and effects of these changes can help us to better understand cancer biology. Citation Format: Lidija D. Vukovic, Bradley A. Stohr, Dan L. Levy. Altering nuclear size impacts cancer cell characteristics in melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2433. doi:10.1158/1538-7445.AM2017-2433