Retinoblastoma protein (Rb) links hypoxia to altered mechanical properties in cancer cells as measured by an optical tweezer

S Khakshour,E J Park,M P Labrecque,F J S Lee,T V Beischlag,H Esmaeilsabzali,M E Cox

doi:10.1038/s41598-017-07947-6

S Khakshour, E J Park + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-07947-6

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Abstract

Hypoxia modulates actin organization via multiple pathways. Analyzing the effect of hypoxia on the biophysical properties of cancer cells is beneficial for studying modulatory signalling pathways by quantifying cytoskeleton rearrangements. We have characterized the biophysical properties of human LNCaP prostate cancer cells that occur in response to loss of the retinoblastoma protein (Rb) under hypoxic stress using an oscillating optical tweezer. Hypoxia and Rb-loss increased cell stiffness in a fashion that was dependent on activation of the extracellular signal-regulated kinase (ERK) and the protein kinase B (AKT)- mammalian target of rapamycin (MTOR) pathways. Pharmacological inhibition of MEK1/2, AKT or MTOR impeded hypoxia-inducible changes in the actin cytoskeleton and inhibited cell migration in Rb-deficient cells conditioned with hypoxia. These results suggest that loss of Rb in transformed hypoxic cancer cells affects MEK1/2-ERK/AKT-MTOR signalling and promotes motility. Thus, the mechanical characterization of cancer cells using an optical tweezer provides an additional technique for cancer diagnosis/prognosis and evaluating therapeutic performance.

Highlights

In cancer and in particular, the tumour microenvironment, hypoxia is a pathological condition in which a significant region of the tumour is deprived of oxygen and is associated with increased risk of metastasis[1, 2]
To determine the effect of hypoxia and loss of retinoblastoma protein (Rb) on the organization of the LNCaP cell cytoskeleton, we examined the normalized amplitude of resultant bead movement (NARBM) in response to an optical tweezer (OT) applied force at a frequency of 1 Hz in short-hairpin-scrambled negative control and short-hairpin-Rb knockdown cells under hypoxic and normoxic conditions (24, 48, 72 h)
Measuring the NARBM in response to the OT applied force demonstrated a time-dependent response in bead displacement that was significantly decreased in short-hairpin-Rb knockdown (shRb) cells exposed to 72 h of hypoxia compared to all other groups (Fig. 1C)

Summary

Introduction

In cancer and in particular, the tumour microenvironment, hypoxia is a pathological condition in which a significant region of the tumour is deprived of oxygen and is associated with increased risk of metastasis[1, 2]. The hypoxic signal mediated by the HIF-1α-ARNT/HIF-1β transcriptional complex[13] induces expression of genes associated with advanced stages of tumour growth and metastasis[14,15,16]. Studying different signalling pathways that modulate actin organization under hypoxia is possible via analyzing the biophysical properties of cancer cells and quantifying cytoskeleton rearrangement[19]. MTOR signalling pathways control actin organization and regulates tumour cell invasion and motility[24]. The hypoxic signal mediated by the HIF-1α-ARNT transcriptional complex causes expression of genes associated with tumour growth and metastasis. Gene ontogeny suggests that the Rb-HIF1 complex mediates the ERK1/2, AKT and NFκB signalling pathways, and perturbations in Rb expression may result in actin impairment and reorganization[16]. In order to quantify the cytoskeletal remodelling and stiffness of control and Rb-depleted LNCaP cells, movement of microbeads that bind to cell surface integrin receptors using an OT were measured and validated

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