Interferon-regulated suprabasin is essential for stress-induced stem-like cell conversion and therapy resistance of human malignancies.

Sona Hubackova,Zdenek Hodny,Miroslav Pribyl,Alena Moudra,Pavel Vodicka,Radka Vaclavikova,Vojtech Tambor,Barbora Salovska,Jiri Svec,Jiri Bartek,Hynek Strnad,Rastislav Dzijak,Lukas Rob,Terezie Imrichova,Lenka Kyjacova

doi:10.1002/1878-0261.12480

Abstract

Radiation and chemotherapy represent standard‐of‐care cancer treatments. However, most patients eventually experience tumour recurrence, treatment failure and metastatic dissemination with fatal consequences. To elucidate the molecular mechanisms of resistance to radio‐ and chemotherapy, we exposed human cancer cell lines (HeLa, MCF‐7 and DU145) to clinically relevant doses of 5‐azacytidine or ionizing radiation and compared the transcript profiles of all surviving cell subpopulations, including low‐adherent stem‐like cells. Stress‐mobilized low‐adherent cell fractions differed from other survivors in terms of deregulation of hundreds of genes, including those involved in interferon response. Exposure of cancer cells to interferon‐gamma but not interferon‐beta resulted in the development of a heterogeneous, low‐adherent fraction comprising not only apoptotic/necrotic cells but also live cells exhibiting active Notch signalling and expressing stem‐cell markers. Chemical inhibition of mitogen‐activated protein kinase/ERK kinase (MEK) or siRNA‐mediated knockdown of extracellular signal‐regulated kinase 1/2 (Erk1/2) and interferon responsible factor 1 (IRF1) prevented mobilization of the surviving low‐adherent population, indicating that interferon‐gamma‐mediated loss of adhesion and anoikis resistance required an active Erk pathway interlinked with interferon signalling by transcription factor IRF1. Notably, a skin‐specific protein suprabasin (SBSN), a recently identified oncoprotein, was among the top scoring genes upregulated in surviving low‐adherent cancer cells induced by 5‐azacytidine or irradiation. SBSN expression required the activity of the MEK/Erk pathway, and siRNA‐mediated knockdown of SBSN suppressed the low‐adherent fraction in irradiated, interferon‐gamma‐ and 5‐azacytidine‐treated cells, respectively, implicating SBSN in genotoxic stress‐induced phenotypic plasticity and stress resistance. Importantly, SBSN expression was observed in human clinical specimens of colon and ovarian carcinomas, as well as in circulating tumour cells and metastases of the 4T1 mouse model. The association of SBSN expression with progressive stages of cancer development indicates its role in cancer evolution and therapy resistance.

Highlights

Over the last decades, genotoxic therapies including chemo- or radiotherapy have been the prevailing modalities used to treat cancer patients
We have shown that exposure of human metastasis-derived prostate cancer (PCa) cell lines to a clinically relevant fractioned ionizing radiation regimen resulted in development of three distinct fIRsurviving populations in vitro (Kyjacova et al, 2015): (1) senescent-like adherent cells with the potential to restart proliferation after the last dose of IR; (2) lowadherent dormant cells with stem-like cell traits and preserved competence to re-attach and restore proliferation; and (3) re-adherent cells derived from the low-adherent fraction with reverted epithelial features and retained tumourigenicity supporting a model of bidirectional interconversion in establishment of tumour cell heterogeneity
Among many deregulated genes detected by cDNA microarray analysis of transcript profiles from anoikisresistant human tumour cells induced by 5-azacytidine or fIR, we identified elevation of gene transcripts regulated by the interferon signalling pathway and interferon gamma (IFNc) itself as being capable of inducing the anoikis-resistant state

Summary

Introduction

Genotoxic therapies including chemo- or radiotherapy have been the prevailing modalities used to treat cancer patients. Accumulating evidence indicates that the resistance of cancer cells to radiotherapy and chemotherapy encompasses several different but partly interlinked mechanisms comprising therapy-induced molecular programs together with the initial intra-tumour genetic and epigenetic heterogeneity (Burrell et al, 2013; Kreso et al, 2013; Swanton, 2012). Recent research has provided evidence that the cellular response to genotoxic stress can contribute to phenotypic reprogramming of cancer cells into more resistant phenotypes. Such process commonly involves induction of epithelial-to-mesenchymal transition (EMT) and occurrence of cancer cells with stem-like characteristics (Ghisolfi et al, 2012; Gomez-Casal et al, 2013; Kyjacova et al, 2015; Skvortsova et al, 2008). Genotoxic therapies increase the migratory and invasive properties of malignant cells (Moncharmont et al, 2014) and alter the tumourassociated microenvironment, collectively promoting metastatic behaviour of treatment-surviving cancer cells (Ruegg et al, 2011)

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