EGFR Inhibition Abrogates Leiomyosarcoma Cell Chemoresistance through Inactivation of Survival Pathways and Impairment of CSC Potential

Giovanni Sette,Vincenzo Canzonieri,Adriana Eramo,Mauro Biffoni,Michele Signore,Ruggero De Maria,Vito D’Andrea,Valentina Salvati,Katia Fecchi,Paola Di Matteo,Lorenzo Memeo,Enrico De Antoni,Cristina Colarossi

doi:10.1371/journal.pone.0046891

Abstract

BackgroundTumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome.Methodology/Principal FindingsWe expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as “tumor spheres” and as “monolayers” in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells.Conclusions/SignificanceEGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients.

Highlights

Soft tissue sarcomas constitute a heterogeneous group of rare tumors, accounting for 1% of adult neoplasias and 10% of pediatric malignancies [1]
In addition to the standard stem cell culture methodology, based on the assumption that the tumorigenic cells in sarcomas should consist in transformed undifferentiated cells of mesenchymal origin, i.e. transformed Mesenchymal Stem Cells (MSC), we evaluated the possibility to isolate these undifferentiated tumor cells in the same culture conditions widely used for non-transformed MSCs [31,32]
CD166, CD44 and CD90 were abundantly present in all three cell culture types analyzed, as expected for these broadly expressed mesenchymal antigens, the MSC-restricted markers CD146 and CD105 were considerably up-regulated in both sarco-spheres (Figure 1A) and adherent MSC-like cultures (Figure 1B) in comparison with LMS cells obtained under standard conditions (Figure 1C)

Summary

Introduction

Soft tissue sarcomas constitute a heterogeneous group of rare tumors, accounting for 1% of adult neoplasias and 10% of pediatric malignancies [1]. Important studies have highlighted a key role of CSC in development, maintenance, metastasis, chemoresistance and relapse of solid tumors, indicating these undifferentiated transformed stem cells as primary targets for more effective anti-cancer therapies [18,19,20,21,22,23,24]. SP cells displayed high tumorigenic potential, while the proportion of SP cells correlated with tumor aggressiveness, suggesting that interfering with the SP phenotype or with other CSC properties could constitute a strategy to counteract soft tissue sarcoma aggressiveness [28,29] These reports highlighted the possibility to investigate the existence and nature of CSCs in different types of soft tissue sarcomas, paving the way for potential identifycation of innovative therapeutic targets for these deadly cancers [29]. Therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome

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Conclusion