William Andrew Yeudall

Cancer stem cells (CSCs) are a small population of cancer cells that endow unique traits of self-renewal, differentiation, tumorigenicity, quiescence and therapeutic resistance, ultimately leading to cancer relapse and poor prognosis. Conventional chemo- and radiotherapies effectively destroy rapidly dividing cancer cells; however, CSCs are largely refractory to these treatments due to special characteristics that include metabolic plasticity, low reactive oxygen species (ROS) levels, apoptosis inhibition and modified DNA repair systems, effectively hijacking the tumour microenvironment and evading host immune surveillance. This review summarises the potential CSC diagnostic methods that can be used in routine clinical investigation for tailoring personalised treatment plans both pre- and post-surgery. In this way, a CSC gene signature, reactivity and load can be estimated and, by using artificial intelligence and machine learning algorithms in a personalised therapeutic regimen, the most effective treatment response can be predicted. Furthermore, we propose incorporation of CSC-targeted therapies in conjugation with conventional chemo- and radiotherapies to eradicate CSCs, thereby eliminating the risk of relapse and therapy failure in cancer patients. This review also describes multiple potential molecular targets that may be used to target CSCs and emphasises the need for further evaluation, in order that they may be applied clinically.

Vimentin is a type III intermediate filament (IF) protein that is induced in a large number of solid tumours. A single cysteine at position 328 in vimentin plays a crucial role in assembly, organisation, and stability of IFs. However, its exact function during epithelial–mesenchymal transition (EMT) and cancer progression has not been investigated. To investigate this, we have transduced wildtype (WT) and C328S vimentin separately in MCF-7 cells that lack endogenous vimentin. The expression of C328-VIM impacted vimentin–actin interactions and induced EMT-like features that include enhanced cell proliferation, migration, and invasion accompanied by reduced cell adhesion when compared to the wildtype cells. Functional transcriptomic studies confirmed the upregulation of EMT and mesenchymal markers, downregulation of epithelial markers, as well as acquisition of signatures associated with cancer stemness (CD56, POU5F1, PROCR, and CD49f), thus transforming MCF-7 cells from oestrogen-positive to triple-reduced (ESR1, PGR, and HER2) status. We also observed a stark increase in the expression of long non-coding RNA, XIST, in MCF-7 cells expressing C328-VIM. Targeting the mutant vimentin or XIST by RNA interference partially reversed the phenotypes in C328-VIM-expressing MCF-7 cells. Furthermore, the introduction of C328-VIM cells into nude mice promoted tumour growth by increasing cancer stemness in an oestrogen-independent manner. Altogether, our studies provide insight into how cysteine 328 in vimentin dictates mechano-transduction signals to remodel actin cytoskeleton and protect against EMT and cancer growth via modulating lncRNA XIST. Therefore, targeting vimentin and/or XIST via RNA interference should be a promising therapeutic strategy for breast cancer treatment.

In advanced metastatic cancers with reduced patient survival and poor prognosis, expression of vimentin, a type III intermediate filament protein is frequently observed. Vimentin appears to suppress epithelial characteristics and augments cell migration but the molecular basis for these changes is not well understood. Here, we have ectopically expressed vimentin in MCF-7 and investigated its genomic and functional implications. Vimentin changed the cell shape by decreasing major axis, major axis angle and increased cell migration, without affecting proliferation. Vimentin downregulated major keratin genes KRT8, KRT18 and KRT19. Transcriptome-coupled GO and KEGG analyses revealed that vimentin-affected genes were linked to either cell–cell/cell-ECM or cell cycle/proliferation specific pathways. Using shRNA mediated knockdown of vimentin in two cell types; MCF-7FV (ectopically expressing) and MDA-MB-231 (endogenously expressing), we identified a vimentin-specific signature consisting of 13 protein encoding genes (CDH5, AXL, PTPRM, TGFBI, CDH10, NES, E2F1, FOXM1, CDC45, FSD1, BCL2, KIF26A and WISP2) and two long non-coding RNAs, LINC00052 and C15ORF9-AS1. CDH5, an endothelial cadherin, which mediates cell–cell junctions, was the most downregulated protein encoding gene. Interestingly, downregulation of CDH5 by shRNA significantly increased cell migration confirming our RNA-Seq data. Furthermore, presence of vimentin altered the lamin expression in MCF-7. Collectively, we demonstrate, for the first time, that vimentin in breast cancer cells could change nuclear architecture by affecting lamin expression, which downregulates genes maintaining cell–cell junctions resulting in increased cell migration.

P53 mutations with single amino acid changes in cancer often lead to dominant oncogenic changes. Here, we have developed a mouse model of gain-of-function (GOF) p53-driven lung cancer utilizing conditionally active LSL p53-R172H and LSL K-Ras-G12D knock-in alleles that can be activated by Cre in lung club cells. Mutation of the p53 transactivation domain (TAD) (p53-L25Q/W26S/R172H) eliminating significant transactivation activity resulted in loss of tumorigenicity, demonstrating that transactivation mediated by or dependent on TAD is required for oncogenicity by GOF p53. GOF p53 TAD mutations significantly reduce phosphorylation of nearby p53 serine 20 (S20), which is a target for PLK3 phosphorylation. Knocking out PLK3 attenuated S20 phosphorylation along with transactivation and oncogenicity by GOF p53, indicating that GOF p53 exploits PLK3 to trigger its transactivation capability and exert oncogenic functions. Our data show a mechanistic involvement of PLK3 in mutant p53 pathway of oncogenesis.

Background: EPS8 is a scaffolding protein that regulates proliferation, actin dynamics and receptor trafficking. Its expression is increased in cancer, enhancing mitogenesis, migration and tumorigenesis. Src phosphorylates EPS8 at four tyrosine residues, although the function is unknown. Here we investigated the pro-oncogenic role of EPS8 tyrosine phosphorylation at Src target sites in HNSCC.Methods: Plasmids expressing EPS8 Src-mediated phosphorylation site mutants (Y485F, Y525F, Y602F, Y774F and all four combined [FFFF]) were expressed in cells containing a normal endogenous level of EPS8. In addition, cells were treated with dasatinib to inhibit Src activity. EPS8 downstream targets were evaluated by western blotting. Wound closure, proliferation, immunofluorescence and tumorgenicity assays were used to investigate the impact of phenylalanine mutations on EPS8 biological functions.Results: FOXM1, AURKA, and AURKB were decreased in cells expressing FFFF- and Y602F-EPS8 mutants, while cells harbouring the Y485F-, Y525F- and Y774F-EPS8 mutants showed no differences compared to controls. Consistent with this, dasatinib decreased the expression of EPS8 targets. Moreover, Y602F- and FFFF-EPS8 mutants reduced mitogenesis and motility. Strikingly though, FFFF- or Y602F-EPS8 mutants actually promoted tumorigenicity compared with control cells.Conclusions: Phosphorylation of EPS8 at Y602 is crucial for signalling to the cell cycle and may provide insight to explain reduced efficacy of dasatinib treatment.

Globally, it has been estimated that there were around 18.1 million new cases of cancer at all sites in 2018, with 9.6 million deaths from the disease (Bray et al., 2018; Ferlay et al., 2019). As such, the mortality rate remains high, in spite of advances in treatment over the past few decades. Cancer is a genetic disease (Hanahan & Weinberg, 2011). Pinpointing key alterations in the cancer genome is, therefore, fundamental for our understanding of disease mechanisms, identifying druggable targets, and improving our knowledge as to why some targeted therapies fail to perform as well as might be expected.

Head and neck squamous cell carcinoma (HNSCC) develops in the mucosal lining of the upper aerodigestive tract, principally as a result of exposure to carcinogens present in tobacco products and alcohol, with oncogenic papillomaviruses also being recognized as etiological agents in a limited proportion of cases. As such, there is considerable scope for prevention of disease development and progression. However, despite multimodal approaches to treatment, tumor recurrence and metastatic disease are common problems, and clinical outcome is unsatisfactory. As our understanding of the genetics and biochemical aberrations in HNSCC has improved, so the development and use of molecularly targeted drugs to combat the disease have come to the fore. In this article, we review molecular mechanisms that alter signal transduction downstream of the epidermal growth factor receptor (EGFR) as well as those that perturb orderly cell cycle progression, such as p53 mutation, cyclin overexpression, and loss of cyclin-dependent kinase inhibitor function. We outline some of the tactics that have been employed to combat the altered biochemistry. These include blockade of the EGFR using humanized monoclonal antibodies such as cetuximab and small molecule tyrosine kinase inhibitors (TKIs) such as erlotinib/gefitinib and subsequent generations of TKIs, restoration of p53 function using MIRA compounds, and inhibition of cyclin-dependent kinase and aurora kinase activity using drugs such as palbociclib and alisertib. Knowledge of the underlying molecular mechanisms may be utilizable in order to predict disease behavior and tailor therapeutic interventions in a more personalized approach to improve clinical response. Use of liquid biopsy, omics platforms, and salivary diagnostics hold promise in this regard.

The functional relationship between apoptosis and autophagy in anticancer drug treatment is extremely complex, and the molecular machinery is obscure. This study aims to investigate the efficacy of CYT997, a novel microtubule-disrupting agent, in head and neck squamous cell carcinomas (HNSCCs) and complete the autophagy-apoptosis puzzle involved in drug action. We report here that CYT997 exhibits anticancer activity by triggering oxidative stress-associated apoptosis in HNSCC cells. Interestingly, upregulation of autophagy by mTOR-dependent pathways appears to have a cytoprotective role in preventing apoptosis by inhibiting CYT997-induced excessively high levels of reactive oxygen species (ROS). Blockade of autophagy by ATG7 depletion or addition of autophagy inhibitor hydroxychloroquine (HCQ) sensitizes HNSCC cells to CYT997 as evidenced by enhanced ROS-associated apoptosis. Moreover, HCQ exhibits a good synergism with CYT997 on induction of apoptosis in HNSCC xenografts without cytotoxicity, suggesting combined treatment of CYT997 with autophagy inhibitors would increase the anticancer efficacy of CYT997. These findings unveil the importance of ROS in crosstalk between autophagy and apoptosis in CYT997 treatment, raising concerns that genetic or pharmacologic blockade of autophagy should be considered in the design of new therapeutics for HNSCC.