Revealing Different Roles of the mTOR-Targets S6K1 and S6K2 in Breast Cancer by Expression Profiling and Structural Analysis

Abstract

Background: The AKT/mTORC1/S6K pathway is frequently overstimulated in breast cancer, constituting a promising therapeutic target. The benefit from mTOR inhibitors varies, likely as a consequence of tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms. The mTORC1 downstream effectors S6K1, S6K2, and 4EBP1 are amplified and overexpressed in breast cancer, associated with a poor outcome and divergent endocrine treatment benefit. S6K1 and S6K2 share high sequence homology, but evidence of partly distinct biological functions is emerging. The aim of this work was to explore possible different roles and treatment target potentials of S6K1 and S6K2 in breast cancer.

Materials and methods: Whole-genome expression profiles were compared for breast tumours expressing high levels of S6K1, S6K2 or 4EBP1, using public datasets, as well as after in vitro siRNA downregulation of S6K1 and/or S6K2 in ZR751 breast cancer cells. In silico homology modelling of the S6K2 kinase domain was used to evaluate its possible structural divergences to S6K1.

Results: Genome expression profiles were highly different in S6K1 and S6K2 high tumours, whereas S6K2 and 4EBP1 profiles showed significant overlaps, both correlated to genes involved in cell cycle progression, among these the master regulator E2F1. S6K2 and 4EBP1 were inversely associated with IGF1 levels, and their prognostic value was shown to be restricted to tumours positive for IGFR and/or HER2. In vitro, S6K1 and S6K2 silencing resulted in upregulation of genes in the mTORC1 and mTORC2 complexes. Isoform-specific silencing also showed distinct patterns, e.g. S6K2 downregulation lead to upregulation of several cell cycle associated genes. Structural analyses of the S6K2 kinase domain showed unique structure patterns, deviating from those of S6K1, facilitating the development of isoform-specific inhibitors. Our data support emerging proposals of distinct biological features of S6K1 and S6K2, suggesting their importance as separate oncogenes and clinical markers, where specific targeting in different breast cancer subtypes could facilitate further individualised therapies.

Fig 1. Spearman’s rank order correlation evaluating associations between S6K1, S6K2, 4EBP1 and E2F1 mRNA expression (continuous values) in three breast cancer cohorts.

Fig 2. Kaplan-Meier curves and multivariate Cox regression of breast cancer survival (BCS) in relation to S6K2 and/or 4EBP1 mRNA, in the van de Vijver, Karolinska and Uppsala patient cohorts respectively.

IGF1R and/or IGF2R and/or HER2 high (a, c, e); IGF1R and IGF2R and HER2 low (b, d, f). The Cox analysis included the following variables: adjuvant chemotherapy treatment, endocrine treatment, lymph node status, tumour size (not available for van de Vijver), ER status and E2F1.

Fig 3. Structural comparison of crystal structures of S6K1 and homology models of S6K2.

(a) Crystal structure of S6K1 (PDB ID: 3A62) with the αC helix highlighted in blue. (b) Overlay of the structure in (a) and a homology model of S6K2 (orange) based on this structure. (c) Crystal structure of S6K1 (PDB ID: 4L3J). (d) Overlay of the structure in (c) and a homology model of S6K2 based on this structure. The colouring is the same as in the previous panels. The images were rendered in PyMol (Schrödinger LLC). See S4 Fig for alternative representations of these images where also key residues are shown.