Focused ultrasound stimulates ER localized mechanosensitive PANNEXIN-1 to mediate intracellular calcium release in invasive cancer cells

Abstract

Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is noninvasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not noninvasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1 however plays a different role in mediating the spread of intercellular calcium waves via ATP release.Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy. FIGURE 3. Treatment of PC-3 cells with 10 PX1 (PANX1 inhibitor) abolishes the normal FUSinduced Ca 2+ oscillation response but uncovers single Ca 2+ transients. (A)Left column, the cells exhibited strong Ca 2+ responses at 20 min after 200 M scrambled peptide application as a control. Center column, cells were stimulated at 20 min after 200 M 10 Panx1 peptide ( 10 PX1) application, and the responses were partly reduced. Right column, 20 min after 2 M Xestospongin C (XC) application, the responses were also partly reduced. Two representative cells were shown in each treatment. (B) Quantitative CRI values of the inhibitor treatments. n=3 (XC), or n=6 (SC, 10 PX1). Error bars, s.e.m., ANOVA, Dunnet's correction, exact p values. (C) Fluorescence patterns in cells that first responded to the stimulus after the treatments. Two representative cells are shown by F/F. (D) Fluorescence patterns in several cells that first responded to the stimulus after the treatments; Scrambled (9 cells), 10 PX1 (5 cells) and XC (6 cells).

ECM dimensionality tunes actin tension to modulate the endoplasmic reticulum and spheroid phenotype

“…Indeed, recent evidence suggests that numerous calcium channels localized to the ER (e.g. Pannexin-1 and Piezo) can be activated by extracellular mechanical stimuli such as uniaxial stretching, focused ultrasound and lasertweezer-traction at the plasma membrane [32][33][34] . We consistently found that cells ligated to an ECM in 2D generate high actin tension and simultaneously develop dysregulated calcium homeostasis.…”

“…A table of the renormalized values of tension is shown in Table S1. A typical value of a0 for the systems we model is a0=10 nm; this value ensures that our choices of c0=0.4-0.8 a0 -1 and e 2 =6.3a0 2 , are primed to model curvature sensing proteins such with BAR and ENTH domains, as justified in previous works 32,33 . With this choice of the value for the scale parameter a0, the characteristic size of the protrusion we model is of diameter 10a0 (as set by the Ising potential), which amounts to 100 nm; this value is consistent with the diameter of membrane protrusions observed in cell studies [W. Guo, private communication, 2015].…”

“…Both these moduli are renormalized during equilibration for different initial conditions of link length. Renormalized tension is calculated by fitting the undulation spectrum of membrane undulations 33 . A table of the renormalized values of tension is shown in Table S1.…”

“…We note that the choice of a0 also sets the range of membrane tensions we model to the range 0-100 µN/m, which is consistent with the range to membrane tension values reported in cellular experiments 66,67 . 32,33,65 .…”

Investigation of Ultrasound-Mediated Intracellular Ca2+ Oscillations in HIT-T15 Pancreatic β-Cell Line

“…We have previously shown that the connexin 43 hemichannel works as a mechanosensor in hMSC that releases ATP to the extracellular space in response to low-intensity ultrasound [18]. In addition, we recently showed that ultrasound-induced ATP release was reduced significantly when the pannexin 1 hemichannel was either inhibited or knocked down in the prostate PC-3 cancer cells [57]. Ultrasound is a very unique modality that can transmit mechanical energy into tissues deep in the body without an invasive procedure.…”