Matrix stiffness-sensitive long noncoding RNA NEAT1 seeded paraspeckles in cancer cells

Abstract

Cancer progression is influenced by changes in the tumor microenvironment, such as the stiffening of the extracellular matrix. Yet our understanding of how cancer cells sense and convert mechanical stimuli into biochemical signals and physiological responses is still limited. The long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), which forms the backbone of subnuclear "paraspeckle" bodies, has been identified as a key genetic regulator in numerous cancers. Here, we investigated whether paraspeckles, as defined by NEAT1 localization, are mechanosensitive. Using tunable polyacrylamide hydrogels of extreme stiffnesses, we measured paraspeckle parameters in several cancer cell lines and observed an increase in paraspeckles in cells cultured on soft (3 kPa) hydrogels compared with stiffer (40 kPa) hydrogels. This response to soft substrate is erased when cells are first conditioned on stiff substrate, and then transferred onto soft hydrogels, suggestive of mechanomemory upstream of paraspeckle regulation. We also examined some well-characterized mechanosensitive markers, but found that lamin A expression, as well as YAP and MRTF-A nuclear translocation did not show consistent trends between stiffnesses, despite all cell types having increased migration, nuclear, and cell area on stiffer hydrogels. We thus propose that paraspeckles may prove of use as mechanosensors in cancer mechanobiology.

FIGURE 1:

Paraspeckle expression on 3 kPa and 40 kPa in MCF10A, U2OS, 143B, and MDA-MB-231 cells. (A) The average number of paraspeckles per nucleus was higher in cancer cell lines cultured on 3 kPa hydrogels compared with 40 kPa hydrogels in U2OS (5.75 vs. 4.28), 143B (9.88 vs. 5.47), and MDA-MB-231 (9.22 vs. 3.44) cell lines. No difference in paraspeckle number was observed in the MCF10A cell line. Treatment of cells cultured on 40 kPa hydrogels with blebbistatin revealed an increase in paraspeckle number in the U2OS (4.28 to 6.00) and MDA-MB-231 (3.44 to 7.65) cell lines. (B) Paraspeckle total area showed the same trend in U2OS (0.021 µm² vs. 0.0094 µm²), 143B (0.017 µm² vs. 0.0082 µm²), and MDA-MB-231 (0.055 µm² to 0.0054 µm²) cell lines but not in the MCF10A cell line. Blebbistatin treatment also resulted in increased paraspeckle area in MCF10A, 143B, and MDA-MB-231 cell lines. (C) Analysis of paraspeckle size revealed that paraspeckles appeared larger in size in cancer cells cultured on 3 kPa hydrogels vs. 40 kPa hydrogels in U2OS (0.0034 µm² vs. 0.0019 µm²), 143B (0.0016 µm² vs. 0.0014 µm²), and MDA-MB-231 (0.0069 µm² vs. 0.0015 µm²) cell lines. Blebbistatin treatment further increased paraspeckle size in all cell lines. (D) Representative images showing paraspeckles (red) in nuclei (blue) taken at 60× magnification. Scale bar = 7.5 µm. Data are shown as mean ± SEM. Numbers of nuclei used in analyses were indicated per bar graph. *, p < 0.05; **, p < 0.01; ***, p < 0.001, and ****, p < 0.0001.

FIGURE 2:

Paraspeckles in MDA-MB-231 cells following conditioning of cells on hydrogels of one stiffness, then transferring cells onto hydrogels of the opposite stiffness, as well as a hydrogel of the same stiffness as a control. (A) The average number of paraspeckles per nucleus and paraspeckle total area remained elevated when cells were conditioned on 3 kPa hydrogels and transferred onto new 3 kPa hydrogels; however, paraspeckle size decreased. When MDA-MB-231 cells were conditioned on 3 kPa hydrogels and transferred onto hydrogels of 40 kPa stiffness, reduced levels of paraspeckle number (5.15 to 3.84), total area (0.011 µm² to 0.0063 µm²), and size (0.002 µm² to 0.001 µm²) were observed. (B) Cells that were conditioned on hydrogels of 40 kPa stiffness and transferred onto hydrogels of the same 40 kPa stiffness, as well as the opposite 3 kPa stiffness, showed no changes in paraspeckle parameters. (C) Representative images showing paraspeckles (red) in nuclei (blue) taken at 60× magnification. Scale bar = 7.5 µm. Data are shown as mean ± SEM. **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

FIGURE 3:

Nuclear area, cell area, and shape of cells revealed that all cell lines appeared morphologically different when cultured on 3 kPa and 40 kPa hydrogels. (A, B) All four cell lines had larger nuclei and cell area when cultured on 40 kPa hydrogels compared with 3 kPa hydrogels. (C) Analysis of form factor, representing the circulatory of cells (1 = a perfect circle) showed that MCF10A, 143B, and MDA-MB-231 cells appeared more circular when cultured on 3 kPa hydrogels. (D) Aspect ratio (X/Y) revealed that 143B cells had a larger X/Y ratio when cultured on stiff substrates. (E) Outlines of nuclear and cell images showing differences in size and morphology of cell cultured on both conditions. Outlines were obtained from images taken at 20× magnification and visualized in multicolor images by CellProfiler using F-actin for cytoplasmic and DAPI for nuclear boundary recognition. Scale bar = 100 µm. Data are shown as mean ± SEM. Numbers of cells used in analyses were indicated per bar graph. *, p < 0.05; **, p < 0.01; and ****, p < 0.0001.

FIGURE 4:

Migration tracking of MCF10A, U2OS, 143B, and MDA-MB-231 cells on 3 kPa vs. 40 kPa hydrogels showed increased migratory trends on stiff substrates. (A) Rose plots corrected to 0,0 showing representative tracks taken by cells cultured on 3 kPa and 40 kPa (n = 20). (B) Total distance traveled confirmed that cells from all cell lines cultured on 40 kPa hydrogels migrated a greater distance (n = 80) compared with cells cultured on 3 kPa hydrogels (n = 80). Data are shown as mean ± SEM. ****, p < 0.0001.

FIGURE 5:

(A) Normalized lamin A expression (n = 3, 100 cells/repeat) revealed that lamin A levels did not change pending on stiffness in MCF10A, U2OS, and 143B cell lines; however, the MDA-MB-231 cell line showed increased normalized lamin A expression in cells cultured on 40 kPa hydrogels compared with soft 3 kPa hydrogels. (B, C) Quantification of YAP and MRTF-A nuclear/cytoplasmic ratio showed no trend between stiffness for both markers in MCF10A, U2OS, 143B, and MDA-MB-231 cell lines (n = 3, 100 cells/repeat). The ratios between markers followed similar trends between cell lines and appeared significantly elevated in the U2OS cell line compared with all other cell lines. (D) Representative images of lamin A, YAP, and MRTF-A staining in all four cell lines at 3 kPa and 40 kPa stiffness taken using confocal microscopy at 20× magnification. (E) Paraspeckle parameters (paraspeckle number, total area, and average size) in MDA-MB-231 cells cultured on 3 kPa, 40 kPa, and glass (E = GPa range) revealed a nonlinear trend between stiffness and paraspeckles. This trend was inverse to the relationship between lamin A and the same stiffness conditions. (F) Investigation of cell proliferation (determined by % confluency changes over time) and paraspeckles in MDA-MB-231 revealed that although cells cultured on 40 kPa and glass exhibited similar proliferation rates, paraspeckle abundance in these two conditions differed. Scale bar = 50 µm. Data are shown as mean ± SEM. *, p < 0.05.