Overexpression of the human antigen R suppresses the immediate paradoxical proliferation of melanoma cell subpopulations in response to suboptimal BRAF inhibition.

Marylise Fernandez,Sylvain Lemeille,Wolf‐Henning Boehncke,Rastine Merat,Christoph Schwärzler,Hedwig Sutterlüty‐Fall

doi:10.1002/cam4.1091

Abstract

Tumor plasticity and the heterogeneous response of melanoma cells to targeted therapies are major limits for the long‐term efficacy of this line of therapy. Targeting tumor plasticity is theoretically possible through the modulation of the expression of RNA‐binding proteins which can affect many different compensatory mechanisms of the adaptive response of malignant cells to targeted therapies. Human antigen R (HuR) is a modulator of gene expression and a transacting factor in the mRNA‐processing machinery used in the cell stress response, and is a potential target for reducing tumor plasticity. In this experiment, we exploit the inherent heterogeneous response of the A375 melanoma line to suboptimal BRAF inhibition as a model of immediate adaptive response. We first observe that HuR overexpression can prevent the heterogeneous response and thus the immediate paradoxical proliferation induced by low‐doses vemurafenib treatment. We then use single‐cell mass cytometry to characterize subpopulations, including those that paradoxically proliferate, based on their proliferation rate and the expression patterns of markers involved in the reversible adaptive resistance to BRAF inhibition and/or recognized as HuR targets involved in cell cycle regulation. Under suboptimal BRAF inhibition, HuR overexpression affects these subpopulations and their expression pattern with contrasting responses depending on their proliferation rate: faster‐proliferating vemurafenib‐sensitive or ‐resistant subpopulations showed higher death tendency and reduced size, and slower‐proliferating subpopulations showed an attenuated resistant expression response and their paradoxical proliferation was inhibited. These observations pave the way to new therapeutic strategies for preventing the heterogeneous response of tumors to targeted therapies.

Highlights

Impressive clinical results have shown that targeted therapies for melanoma, that is, BRAF and MEK inhibitors, can efficiently treat highly mutagenic solid malignancies by blocking critical cell survival pathways
We have repeatedly observed that some BRAF-mutated sensitive melanoma cell lines treated with low-dose suboptimal vemurafenib (i.e., 20 nmol/L and up to 100 nmol/L depending on the cell lines) may paradoxically proliferate
C-Myc staining was not discriminative regarding the paradoxical proliferation of any of the subpopulations, but a higher expression level was detected in aHt cells, mainly in Sp3 compared with aHnt cells. These observations indicate that Human antigen R (HuR) overexpression can suppress the immediate heterogeneous response to low- dose BRAF inhibition and the subsequent paradoxical proliferation that occurs in some subpopulations of the sensitive A375 melanoma cell line

Summary

Introduction

Impressive clinical results have shown that targeted therapies for melanoma, that is, BRAF and MEK inhibitors, can efficiently treat highly mutagenic solid malignancies by blocking critical cell survival pathways. HuR Blocks Paradoxical Growth of Melanoma Cells prevention of reversible nongenetic adaptive resistance is most likely possible, but any efficient strategy to prevent the emergence of compensatory mechanisms likely needs to have a simultaneous effect on multiple major nodes within the cancer signaling network [7]. Ubiquitously expressed [14], HuR presence within the mRNA-processing machinery seems less obligatory than other cancer- deregulated RBPs like, for example, eIF4E. This variability in its presence is used in the cell stress response to many environmental changes such as UV radiation [15] and makes HuR an excellent candidate for modulating cell plasticity and heterogeneity. Using single-c ell mass cytometry, we characterize the expression profile and behavior of various cell subpopulations within this cell line toward the BRAF inhibitor vemurafenib and under the effect of HuR overexpression, and observe that when overexpressed, HuR can overcome the emergence of paradoxically proliferative subpopulations

Methods

Results

Conclusion