Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells ...NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.
CK2 is a kinase family that is known to phosphorylate many cellular proteins and is frequently upregulated in cancer. CK2 inhibitors are currently in clinical trials, making CK2 a newly appreciated ...therapeutic target for cancer treatment. Cancers, amongst other diseases, often display dysregulated protein synthesis, which is a process wherein mRNAs are translated to proteins, also referred to as mRNA translation. Along with increased global protein synthesis, cancer cells can alter the translatome (a transcriptome-wide collection of mRNAs that is actively translated in the cell at a given time), to favour the synthesis of survival-, proliferation-, metabolism- and oncogenesis-related proteins. Such changes in translation are a result of alterations in either the expression of translation factors or pathways that dictate their activity. CK2 has been implicated in the regulation of several cellular processes including translation, where it coordinates the two rate-limiting steps of the initiation phase, namely, eIF4F complex assembly and recycling of the ternary complex. However, the role of CK2 in regulating mRNA translation is still not fully understood. Considering that CK2 has been implicated in several cancers and biological processes that drive malignant tumour formation, it is crucial to understand the role of CK2 in translation. Herein, we determined the effects of CK2 inhibition on the translatome and ensuing cancer-related phenotypes. This revealed a previously unappreciated role of CK2 in regulating translation in neoplasia, laying the foundation to further explore this relationship.
Specificity is a limiting factor when using small-molecule inhibitors to study protein kinase signalling. Since inhibitor-resistant kinase mutants (i.e., drug-resistant alleles) remain active in the ...presence of inhibitor, they facilitate validation of on-target effects. By combining an inhibitor-resistant kinase mutant with mass spectrometry-based phosphoproteomics, we previously devised a systematic strategy for reliable identification and validation of CSNK2 substrates. In this study, we use the same strategy to evaluate the selectivity of CX-4945, a clinical stage CSNK2 inhibitor, and SGC-CK2-1, a chemical probe selectively targeting CSNK2. Human osteosarcoma (U2OS) cells expressing exogenous wild-type CSNK2A1 (WT) or an inhibitor-resistant triple mutant (TM, V66A/H160D/I174A) were treated with CX-4945 or SGC-CK2-1 prior to analysis using triple SILAC (phospho)proteomics. The minority of phosphosites, 15% at 4 h and 5% at 24 h, that were significantly downregulated in response to CX-4945 treatment were determined to be CSNK2A1-dependent. By comparison, the majority of phosphosites, >55% at both 4 and 24 h, that were significantly downregulated in response to SGC-CK2-1 were identified as CSNK2A1-dependent. This indicates that SGC-CK2-1 exhibits significantly greater selectivity towards CSNK2A1 than CX-4945. Notably, utilization of SGC-CK2-1 in cells expressing CSNK2A1-TM enabled the identification of >300 CSNK2A1-dependent phosphosites. Overall, this study highlights the utility of exploiting highly selective chemical probes together with inhibitor-resistant kinase mutants to facilitate identification of bona fide kinase substrates.
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