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. 2012 Nov;17(6):765-78.
doi: 10.1007/s12192-012-0349-z. Epub 2012 Jul 13.

Heat stress induces epithelial plasticity and cell migration independent of heat shock factor 1

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Heat stress induces epithelial plasticity and cell migration independent of heat shock factor 1

B J Lang et al. Cell Stress Chaperones. 2012 Nov.

Abstract

Current cancer therapies including cytotoxic chemotherapy, radiation and hyperthermic therapy induce acute proteotoxic stress in tumour cells. A major challenge to cancer therapeutic efficacy is the recurrence of therapy-resistant tumours and how to overcome their emergence. The current study examines the concept that tumour cell exposure to acute proteotoxic stress results in the acquisition of a more advanced and aggressive cancer cell phenotype. Specifically, we determined whether heat stress resulted in an epithelial-to-mesenchymal transition (EMT) and/or the enhancement of cell migration, components of an advanced and therapeutically resistant cancer phenotype. We identified that heat stress enhanced cell migration in both the lung A549, and breast MDA-MB-468 human adenocarcinoma cell lines, with A549 cells also undergoing a partial EMT. Moreover, in an in vivo model of thermally ablated liver metastases of the mouse colorectal MoCR cell line, immunohistological analysis of classical EMT markers demonstrated a shift to a more mesenchymal phenotype in the surviving tumour fraction, further demonstrating that thermal stress can induce epithelial plasticity. To identify a mechanism by which thermal stress modulates epithelial plasticity, we examined whether the major transcriptional regulator of the heat shock response, heat shock factor 1 (HSF1), was a required component. Knockdown of HSF1 in the A549 model did not prevent the associated morphological changes or enhanced migratory profile of heat stressed cells. Therefore, this study provides evidence that heat stress significantly impacts upon cancer cell epithelial plasticity and the migratory phenotype independent of HSF1. These findings further our understanding of novel biological downstream effects of heat stress and their potential independence from the classical heat shock pathway.

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Figures

Fig. 1
Fig. 1
Heat shock treatment increases the migratory capacity of cancer cell lines. a Activation of the HSR following heat shock in both the A549 and MDA-MB-468 cell lines as shown by HSF1 phosphorylation at serine326 and the induction of HSPH1 (HSP105), HSPA1A (HSP70-1) and HSPB1 (HSP27) expression. b Heat shock of A549 (n = 2) and MDA-MB-468 (n = 3) cells induces a significant increase in chemotactic cell migration following recovery. Chemoattractants include 0.1 % bovine serum albumin (BSA) as a background migration control and 10 ng/ml epidermal growth factor (EGF), ***p < 0.001
Fig. 2
Fig. 2
Heat shock induces alterations in EMT marker expression, localisation and cell morphology in A549 lung adenocarcinoma cells. a Cell scattering and change in morphology in heat-shocked A549 cell following 24 h of recovery, no distinct change is seen in the MDA-MB-468 cells, scale bar 100 μm. b Heat shock reduces the protein expression of the epithelial marker E-cadherin following 24 h of recovery in A549 cells. Densitometry represents the average fold change ±SD normalised to the loading control of actin (n = 3). c RT-qPCR showing downregulation of expression of the E-cadherin (CDH1) gene following 4 h of recovery. The graph represents fold change ±SD normalised to the RPL32 control gene (n = 3). d Heat shock induces altered levels and localisation of E-cadherin in A549 cells following 24-h recovery. The graph represents quantified E-cadherin distribution across the cell, average ±SEM, (n = 3), scale bar 25 μm, *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
Altered EMT marker expression in residual tumour fraction after TA treatment. Mice with colorectal liver metastases had two selected tumours thermally ablated. a and b Serial sections of formalin-fixed control tissues stained with antibodies against E-cadherin and Zeb1, respectively. c and d Serial sections of formalin fixed thermally ablated tumour tissues stained with antibodies to E- cadherin and Zeb1 respectively. N = tumour necrotic area, TN = thermally induced necrotic area, T = tumour, L = liver. Scale bar 250 μm, insert scale bar 50 μm
Fig. 4
Fig. 4
Heat shock induces morphological change, altered EMT maker expression and enhanced chemomigration independent of HSF1. a Stable expression of targeted HSF1 shRNAmirs in A549 cells significantly reduces HSF1 protein expression and prevents induction of HSP expression following heat shock when compared to shRNAmir scramble control cells. b Heat shock induces cell dissociation independently of HSF1. c Heat shock induces altered expression of epithelial marker E-cadherin in HSF1 knockdown A549 cells, densitometry represents average fold change ±SD normalised to the scramble ctrl, (n = 3). d Heat shock with recovery enhances chemotactic cell migration in the A549 cell line independent of HSF1 (n = 2). Chemoattractants used are indicated at the top and were 0.1 % bovine serum albumin (BSA) and 10 ng/ml epidermal growth factor (EGF). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
MG132 treatment induces morphological changes and enhanced chemomigration independently of HSF1 in the A549 cell line. a Optimisation of MG132 treatment that induces a HSR in A549 cells. b Treatment of A549 cells with 2 μM MG132 for 5 h followed with 24-h recovery induces enhanced chemotactic cell migration (n = 2). c MG132 treatment induces disassociation of cells in standard tissue culture scale bar = 100 μm, with no significant difference in EMT marker expression (n = 2). d HSP expression is not induced by MG132 treatment in A549 cells expressing HSF1 shRNAmir. e Cell lines stably expressing HSF1 targeted shRNAmirs underwent morphological changes following MG132 treatment, scale bars 100 μm. f MG132 treatment with recovery enhanced chemotactic cell migration in the A549 cell line, independently of HSF1 (n = 2). Chemoattractants used in the experiment are labelled at the top and are 0.1 % bovine serum albumin (BSA) and 10 ng/ml epidermal growth factor (EGF). ***p < 0.001

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