Licochalcone A inhibits EGFR signalling and translationally suppresses survivin expression in human cancer cells

Dysfunction of epidermal growth factor receptor (EGFR) signalling plays a critical role in the oncogenesis of non–small-cell lung cancer (NSCLC). Here, we reported the natural product, licochalcone A, exhibited a profound anti-tumour efficacy through directly targeting EGFR signalling. Licochalcone A inhibited in vitro cell growth, colony formation and in vivo tumour growth of either wild-type (WT) or activating mutation EGFR-expressed NSCLC cells. Licochalcone A bound with L858R single-site mutation, exon 19 deletion, L858R/T790M mutation and WT EGFR ex vivo, and impaired EGFR kinase activity both in vitro and in NSCLC cells. The in silico docking study further indicated that licochalcone A interacted with both WT and mutant EGFRs. Moreover, licochalcone A induced apoptosis and decreased survivin protein robustly in NSCLC cells. Mechanistically, we found that treatment with licochalcone A translationally suppressed survivin through inhibiting EGFR downstream kinases ERK1/2 and Akt. Depletion of the translation initiation complex by eIF4E knockdown effectively inhibited survivin expression. In contrast, knockdown of 4E-BP1 showed the opposite effect and dramatically enhanced survivin protein level. Overall, our data indicate that targeting survivin might be an alternative strategy to sensitize EGFR-targeted therapy.     

Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.     

Rhodobacter sphaeroides: complexity in chemotactic signalling

Most bacteria have much more complex chemosensory systems than those of the extensively studied Escherichia coli. Rhodobacter sphaeroides, for example, has multiple homologues of the E. coli chemosensory proteins. The roles of these homologues have been extensively investigated using a combination of deletion, subcellular localization and phosphorylation assays. These studies have shown that the homologues have specific roles in the sensory pathway, and they differ in their cellular localization and interactions with other components of the pathway. The presence of multiple chemosensory pathways might enable bacteria to tune their tactic responses to different environmental conditions.     

ADAMs: key components in EGFR signalling and development

ADAM (a disintegrin and metalloprotease) proteins are membrane-anchored metalloproteases that process and shed the ectodomains of membrane-anchored growth factors, cytokines and receptors. ADAMs also have essential roles in fertilization, angiogenesis, neurogenesis, heart development and cancer. Research on ADAMs and their role in protein ectodomain shedding is emerging as a fertile ground for gathering new insights into the functional regulation of membrane proteins.     

Proteomics moves from expression to turnover: update and future perspective

Proteomics is a rapidly developing discipline that seeks to understand the role of proteins in the wider biological context. In order to take a holistic view of a biological system, it is vital that we can elucidate the dynamics of the proteome. In this article, we have outlined the recent advances in experimental strategies for measuring protein synthesis and degradation on a proteome-wide scale. The application of mass spectrometry and non-mass spectrometric-based approaches in this field of research has been discussed. The article also explores the challenges associated with these types of analyses and the development of appropriate bioinformatic resources for interrogating the complex datasets that are generated.     

Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation

Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules from opposite spindle poles. The spindle-assembly checkpoint is a surveillance mechanism in eukaryotes that inhibits anaphase until all chromosomes have bioriented. In present models, the recruitment of the spindle-assembly checkpoint protein Mad2, through Mad1, to non-bioriented kinetochores is needed to stop cell-cycle progression. However, it is unknown whether Mad1-Mad2 targeting to kinetochores is sufficient to block anaphase. Furthermore, it is unclear whether regulators of biorientation (for example, Aurora kinases) have checkpoint functions downstream of Mad1-Mad2 recruitment or whether they act upstream to quench the primary error signal. Here, we engineered a Mad1 construct that localizes to bioriented kinetochores. We show that the kinetochore localization of Mad1 is sufficient for a metaphase arrest that depends on Mad1-Mad2 binding. By uncoupling the checkpoint from its primary error signal, we show that Aurora, Mps1 and BubR1 kinases, but not Polo-like kinase, are needed to maintain checkpoint arrest when Mad1 is present on kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1-Mad2 to kinetochores, may be signalled not only through Mad2 template dynamics, but also through the activity of widely conserved kinases, to ensure the fidelity of cell division.     

EGFR pathway targeting drugs in head and neck cancer in the era of immunotherapy

Receptor tyrosine kinases (RTKs) are cell surface receptors that bind growth factor ligands and initiate cellular signaling. Of the 20 classes of RTKs, 7 classes, I-V, VIII, and X, are linked to head and neck cancers (HNCs). We focus on the first class of RTK, epidermal growth factor receptor (EGFR), as it is the most thoroughly studied class. EGFR overexpression is observed in 20% of tumors, and expression of EGFR variant III is seen in 15% of aggressive chemoradiotherapy resistant HNCs. Currently, the EGFR monoclonal antibody (mAb) cetuximab is the only FDA approved RTK-targeting drug for the treatment of HNCs. Clinical trials have also included EGFR mAbs, with tyrosine kinase inhibitors, and small molecule inhibitors targeting the EGFR, MAPK, and mTOR pathways. Additionally, Immunotherapy has been found to be effective in 15 to 20% of patients with recurrent or metastatic HNC as a monotherapy. Thus, attempts are underway for the combinatorial treatment of immunotherapy and EGFR mAbs to determine if the recruitment of immune cells in the tumor microenvironment can overcome EGFR resistance.     

Autophagic targeting of Src promotes cancer cell survival following reduced FAK signalling

Here we describe a mechanism that cancer cells use to survive when flux through the Src/FAK pathway is severely perturbed. Depletion of FAK, detachment of FAK-proficient cells or expression of non-phosphorylatable FAK proteins causes sequestration of active Src away from focal adhesions into intracellular puncta that co-stain with several autophagy regulators. Inhibition of autophagy results in restoration of active Src at peripheral adhesions, and this leads to cancer cell death. Autophagic targeting of active Src is associated with a Src-LC3B complex, and is mediated by c-Cbl. However, this is independent of c-Cbl E3 ligase activity, but is mediated by an LC3-interacting region. Thus, c-Cbl-mediated autophagic targeting of active Src can occur in cancer cells to maintain viability when flux through the integrin/Src/FAK pathway is disrupted. This exposes a previously unrecognized cancer cell vulnerability that may provide a new therapeutic opportunity.     

Ring finger protein 12 activates AKT signalling to promote the progression of liver cancer by interacting with EGFR

Liver cancer is one of the most common solid tumours, and ranks as the third leading cause of cancer-associated mortality around the world. This study has linked RNF12 to the pathogenesis of liver cancer. Based on the analysis of patient samples and database data, high RNF12 expression was found in liver cancer, in correlation with worse clinicopathological features and a poor prognosis. Meantime, RNF12 could promote the progression of liver cancer in vitro and in vivo. Mechanistically, RNF12 could interact with EGFR and decrease the internalization of EGFR to activate EGF/EGFR signalling. In addition, PI3K-AKT signalling takes part in the regulation of liver cancer cell proliferation and migration of RNF12. And AKT inhibitor MK2206 could reverse RNF12-mediated cellular proliferation and migration in liver cancer. The possibility of the physical interaction between RNF12 and EGFR might lay a foundation to develop intervention strategies for liver cancer prevention and therapy.     

EGFR signaling in breast cancer: Bad to the bone

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. This family includes EGFR/ErbB1/HER1, ErbB2/HER2/Neu ErbB3/HER3, and ErbB4/HER4. For many years it was believed that EGFR plays a minor role in the development and progression of breast malignancies. However, recent findings have led investigators to revisit these beliefs. Here we will review these findings and propose roles that EGFR may play in breast malignancies. In particular, we will discuss the potential roles that EGFR may play in triple-negative tumors, resistance to endocrine therapies, maintenance of stem-like tumor cells, and bone metastasis. Thus, we will propose the contexts in which EGFR may be a therapeutic target.     

Robustness and complexity co-constructed in multimodal signalling networks

In animal communication, signals are frequently emitted using different channels (e.g. frequencies in a vocalization) and different modalities (e.g. gestures can accompany vocalizations). We explore two explanations that have been provided for multimodality: (i) selection for high information transfer through dedicated channels and (ii) increasing fault tolerance or robustness through multichannel signals. Robustness relates to an accurate decoding of a signal when parts of a signal are occluded. We show analytically in simple feed-forward neural networks that while a multichannel signal can solve the robustness problem, a multimodal signal does so more effectively because it can maximize the contribution made by each channel while minimizing the effects of exclusion. Multimodality refers to sets of channels where within each set information is highly correlated. We show that the robustness property ensures correlations among channels producing complex, associative networks as a by-product. We refer to this as the principle of robust overdesign. We discuss the biological implications of this for the evolution of combinatorial signalling systems; in particular, how robustness promotes enough redundancy to allow for a subsequent specialization of redundant components into novel signals.     

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFR^tyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFR^tyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFR^tyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFR^tyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.Non-small-cell lung cancer (NSCLC) accounts for ∼80% of lung cancer subtypes and is the leading cause of cancer-related death worldwide.¹ In recent years, molecular characterization of NSCLC has reached an unprecedented detail and has allowed segregating NSCLC into discrete molecular subgroups, characterized by specific oncogenic drivers, such as epidermal growth factor receptor (EGFR), BRAF, KRAS, epidermal growth factor receptor 2 (HER2) mutations, MET amplification and anaplastic lymphoma kinase gene rearrangements (ALK).^{2, 3} Consequently, the understanding of NSCLC biology has brought two new classes of targeted agents into the clinical setting: EGFR tyrosine kinase inhibitors (TKIs) and ALK inhibitors.^{4, 5} In particular, clinical trials have shown that NSCLC patients whose tumors harbor sensitizing EGFR mutations significantly benefit from the upfront use of an EGFR TKI, rather than conventional chemotherapy.^{6, 7, 8, 9, 10, 11} Although licensed for clinical use in chemotherapy-pretreated patients, regardless of EGFR mutational status, the EGFR TKI erlotinib has limited efficacy when compared with standard chemotherapy in patients with WT-EGFR NSCLC.^{12, 13, 14}However, a fraction of patients on erlotinib treatment may achieve clinically significant objective responses and prolonged disease control, despite the lack of detectable EGFR mutations.¹⁵ Nevertheless, no biomarker investigated so far was felt sufficiently robust to select for the use of erlotinib in the maintenance or refractory setting.¹⁶ Thus, it would be crucial to identify molecular predictors of TKI sensitivity in EGFR wild-type (WT) tumors in order to prospectively select the subgroup of patients who may benefit from erlotinib therapy. Moreover, EGFR TKIs have also shown a modest therapeutic effect in lung squamous cell carcinoma (SCC), where EGFR mutations are very rare and patients have limited therapeutic options in the maintenance and relapsed settings.^{16, 17, 18, 19, 20}Even more importantly, in order to obtain meaningful clinical responses it is crucial to effectively target the population of cells that are able to escape treatment and maintain the growth of a resistant tumor.²¹ Cancer stem cells (CSCs) have been in fact identified within most solid tumors, including lung tumors, and are associated with increased resistance to therapies.^{22, 23, 24, 25, 26, 27, 28, 29, 30} Thus, the efficacy of innovative therapeutic strategies should be validated against these more aggressive, tumor-maintaining cells.^{23, 27, 31} Importantly, TKI response has never been determined at the level of the tumor-maintaining CSCs. Thus, we investigated erlotinib response of EGFR mutation-negative lung cancer stem cells (LCSCs) and LCSC-based xenografts with the attempt to evaluate their sensitivity to the drug and correlate it with their molecular pattern in order to identify potential biomarkers predictive of erlotinib response in a WT-EGFR context at the CSC level.     

The Notch signalling system: recent insights into the complexity of a conserved pathway

Notch signalling links the fate of one cell to that of an immediate neighbour and consequently controls differentiation, proliferation and apoptotic events in multiple metazoan tissues. Perturbations in this pathway activity have been linked to several human genetic disorders and cancers. Recent genome-scale studies in Drosophila melanogaster have revealed an extraordinarily complex network of genes that can affect Notch activity. This highly interconnected network contrasts our traditional view of the Notch pathway as a simple linear sequence of events. Although we now have an unprecedented insight into the way in which such a fundamental signalling mechanism is controlled by the genome, we are faced with serious challenges in analysing the underlying molecular mechanisms of Notch signal control.     

Cell adhesion and EGFR activation regulate EphA2 expression in cancer

EphA2 is frequently overexpressed in cancer, and increasing amounts of evidence show that EphA2 contributes to multiple aspects of the malignant character including angiogenesis and metastasis. Several aspects of the regulation and functional significance of EphA2 expression in cancer are still largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability. These effects were however abolished by activation of the EGF-receptor ligand system favoring Ras/MAPK signaling and cell proliferation. Based on our results, we propose a regulatory mechanism where cell adhesion induces EGFR kinase activation and EphA2 expression; and where the effect of ephrinA1 mediated reduction in cell viability by inhibiting EphA2 expression is overruled by activated EGFR in human cancer cells.