Extracellular Vesicles and Epidermal Growth Factor Receptor Activation: Interplay of Drivers in Cancer Progression

Extracellular vesicles (EVs) are of great interest to study the cellular mechanisms of cancer development and to diagnose and monitor cancer progression. EVs are a highly heterogeneous population of cell derived particles, which include microvesicles (MVs) and exosomes (EXOs). EVs deliver intercellular messages transferring proteins, lipids, nucleic acids, and metabolites with implications for tumour progression, invasiveness, and metastasis. Epidermal Growth Factor Receptor (EGFR) is a major driver of cancer. Tumour cells with activated EGFR could produce EVs disseminating EGFR itself or its ligands. This review provides an overview of EVs (mainly EXOs and MVs) and their cargo, with a subsequent focus on their production and effects related to EGFR activation. In particular, in vitro studies performed in EGFR-dependent solid tumours and/or cell cultures will be explored, thus shedding light on the interplay between EGFR and EVs production in promoting cancer progression, metastases, and resistance to therapies. Finally, an overview of liquid biopsy approaches involving EGFR and EVs in the blood/plasma of EGFR-dependent tumour patients will also be discussed to evaluate their possible application as candidate biomarkers

Vie di segnalazione staminali nel carcinoma mammario a fenotipo basale: il ruolo di beta-catenina e di HIF-1alpha

Basal-like tumor is an aggressive breast carcinoma subtype that displays an expression signature similar to that of the basal/myoepithelial cells of the breast tissue. Basal-like carcinoma are characterized by over-expression of the Epidermal Growth Factor receptor (EGFR), high frequency of p53 mutations, cytoplasmic/nuclear localization of beta-catenin, overexpression of the Hypoxia inducible factor (HIF)-1alpha target Carbonic Anhydrase isoenzime 9 (CA9) and a gene expression pattern similar to that of normal and cancer stem cells, including the over-expression of the mammary stem cell markers CD44. In this study we investigated the role of p53, EGFR, beta-catenin and HIF-1alpha in the regulation of stem cell features and genes associated with the basal-like gene expression profile. The findings reported in this investigation indicate that p53 inactivation in ductal breast carcinoma cells leads to increased EGFR mRNA and protein levels. In our experimental model, EGFR overexpression induces beta-catenin cytoplasmatic stabilization and transcriptional activity and, by that, leads to increased aggressive features including mammosphere (MS) forming and growth capacity, invasive potential and overexpression of the mammary stem cell gene CD44. Moreover we found that EGFR/beta-catenin axis promotes hypoxia survival in breast carcinoma cells via increased CA9 expression. Indeed beta-catenin positively regulates CA9 expression upon hypoxia exposure. Interestingly we found that beta-catenin inhibits HIF-1alpha transcriptional activity. Looking for the mechanism, we found that CA9 expression is promoted by HIF-1alpha and cytoplasmatic beta-catenin further increased it post-transcriptionally, via direct mRNA binding and stabilization. These data reveal a functional beta-catenin/HIF-1alpha interplay among hallmarks of basal-like tumors and unveil a new functional role for cytoplasmic beta-catenin in the phenotype of such tumors. Therefore it can be proposed that the interplay here described among EGFR/beta-catenin and HIF-1alpha may play a role in breast cancer stem cell survival and function

Glucocorticoid Receptor Modulates EGFR Feedback upon Acquisition of Resistance to Monoclonal Antibodies

Evidences of a crosstalk between Epidermal Growth Factor Receptor (EGFR) and Glucocorticoid Receptor (GR) has been reported, ranging from the modulation of receptor levels or GR mediated transcriptional repression of EGFR target genes, with modifications of epigenetic markers. The present study focuses on the involvement of EGFR positive and negative feedback genes in the establishment of cetuximab (CTX) resistance in metastatic Colorectal Cancer (CRC) patients. We evaluated the expression profile of the EGFR ligands TGFA and HBEGF, along with the pro-inflammatory cytokines IL-1B and IL-8, which were previously reported to be negatively associated with monoclonal antibody response, both in mice and patient specimens. Among EGFR negative feedback loops, we focused on ERRFI1, DUSP1, LRIG3, and LRIG1. We observed that EGFR positive feedback genes are increased in CTX-resistant cells, whereas negative feedback genes are reduced. Next, we tested the expression of these genes in CTX-resistant cells upon GR modulation. We unveiled that GR activation leads to an increase in ERRFI1, DUSP1, and LRIG1, which were shown to restrict EGFR activity, along with a decrease in the EGFR activators (TGFA and IL-8). Finally, in a cohort of xenopatients, stratified for response to cetuximab, we observed an inverse association between the expression level of LRIG1 and CRC progression upon CTX treatment. Our model implies that combining GR modulation to EGFR inhibition may yield an effective treatment strategy in halting cancer progression

A Novel Role for the Interleukin-1 Receptor Axis in Resistance to Anti-EGFR Therapy

Cetuximab (CTX) is a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), commonly used to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, objective remissions occur only in a minority of patients and are of short duration, with a population of cells surviving the treatment and eventually enabling CTX resistance. Our previous study on CRC xenopatients associated poor response to CTX with increased abundance of a set of pro-inflammatory cytokines, including the interleukins IL-1A, IL-1B and IL-8. Stemming from these observations, our current work aimed to assess the role of IL-1 pathway activity in CTX resistance. We employed a recombinant decoy TRAP IL-1, a soluble protein combining the human immunoglobulin Fc portion linked to the extracellular region of the IL-1-receptor (IL-1R1), able to sequester IL-1 directly from the medium. We generated stable clones expressing and secreting a functional TRAP IL-1 into the culture medium. Our results show that IL-1R1 inhibition leads to a decreased cell proliferation and a dampened MAPK and AKT axes. Moreover, CRC patients not responding to CTX blockage displayed higher levels of IL-1R1 than responsive subjects, and abundant IL-1R1 is predictive of survival in patient datasets specifically for the consensus molecular subtype 1 (CMS1). We conclude that IL-1R1 abundance may represent a therapeutic marker for patients who become refractory to monoclonal antibody therapy, while inhibition of IL-1R1 by TRAP IL-1 may offer a novel therapeutic strategy

Beta-Catenin/HuR Post-Transcriptional Machinery Governs Cancer Stem Cell Features in Response to Hypoxia

<div><p>Hypoxia has been long-time acknowledged as major cancer-promoting microenvironment. In such an energy-restrictive condition, post-transcriptional mechanisms gain importance over the energy-expensive gene transcription machinery. Here we show that the onset of hypoxia-induced cancer stem cell features requires the beta-catenin-dependent post-transcriptional up-regulation of CA9 and SNAI2 gene expression. In response to hypoxia, beta-catenin moves from the plasma membrane to the cytoplasm where it binds and stabilizes SNAI2 and CA9 mRNAs, in cooperation with the mRNA stabilizing protein HuR. We also provide evidence that the post-transcriptional activity of cytoplasmic beta-catenin operates under normoxia in basal-like/triple-negative breast cancer cells, where the beta-catenin knockdown suppresses the stem cell phenotype <i>in vitro</i> and tumor growth <i>in vivo</i>. In such cells, we unravel the generalized involvement of the beta-catenin-driven machinery in the stabilization of EGF-induced mRNAs, including the cancer stem cell regulator IL6. Our study highlights the crucial role of post-transcriptional mechanisms in the maintenance/acquisition of cancer stem cell features and suggests that the hindrance of cytoplasmic beta-catenin function may represent an unprecedented strategy for targeting breast cancer stem/basal-like cells.</p> </div

Beta-catenin stabilizes CA9 and SNAI2 mRNAs through direct binding and facilitating the shift of HuR/mRNA complexes to the ribosomal compartment.

<p><b>A</b>, CA9 and SNAI2 3’UTR-luciferase reporter (CA9-3’UTR-Luc and SNAI2-3’UTR-Luc) assay in ctrl/shBeta MCF7 cells exposed to Nor/1%pO₂ conditions; <b>B</b>, CA9 and SNAI2 3’UTR-luciferase reporter assay in ctrl/shBeta MDA-MB-468 cells; Schematic diagram of the CA9 or SNAI2 3’UTR luciferase encoding vectors carrying either CA9 or SNAI2 3’UTR sequences inserted between the luciferase coding sequence and the polyadenylation site; <b>C</b>, Quantitative CA9 and SNAI2 mRNA immunoprecipitation assay with mouse IgG/beta-catenin antibody in 1%pO₂ MCF7 cells, MDA-MB-468 and MDA-MB-231 cells; <b>D</b>, WB analysis of cytoplasm pre-ribosomal (PRF) and 40S ribosomal (40S) cytoplasmic fractions of ctrl/shBeta MCF7 upon exposure to Nor/1%pO₂ conditions; total beta-catenin protein levels are reported in Figure 2A and S5A; E, Real-Time PCR analysis of CA9 mRNA levels in PRF/40S cytoplasmic fractions of 1%pO₂ exposed ctrl/shBeta MCF7, MDA-MB-468 and MDA-MB-231 cells; <b>F</b>, WB of beta-catenin and HuR protein levels in ctrl/beta-wt and in ctrl/shBeta MCF7 cells; <b>G</b>, WB analysis of HuR in PRF and 40S cytoplasmic fractions of ctrl/shBeta MCF7 cells exposed to Nor/1%pO₂; note that total HuR protein levels of Nor/1%pO₂ MCF7 cells refer to Figure S5D; H, Schematic representation of the cytoplasmic beta-catenin/HuR post-transcriptional machinery in the regulation of CA9 and SNAI2 mRNAs, via stabilization, direct binding and shuttling to the ribosomal compartment. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

Beta-catenin maintains the stem/progenitor cell pool in normoxia, independently of its nuclear transcriptional activity.

<p><b>A</b>, CA9 and SNAI2 Real Time PCR analysis and CA9-luc and SNAI2-Luc promoter activity in adherent MCF7 cells and MCF7-MS; <b>B</b>, WB analysis of SNAI2 and CA9 in MCF7-MS; <b>C</b>, beta-catenin IF analysis in adherent MCF7 cells and MCF7-MS; <b>D</b>, TOPFLASH assay and WB analysis of beta-catenin in adherent MCF7 cells and MCF7-MS. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p

Hypoxia induces CA9 and SNAI2 expression via HIF1-alpha dependent mRNA production and beta-catenin dependent stabilization.

<p><b>A</b>, HIF-1alpha transcriptional reporter (HRE-Luc) assay in MCF7 cells transfected with wild type beta-catenin (Beta-wt) under Nor/1%pO₂ conditions or in combination with HIF1-alpha (HIF1a) encoding vector; <b>B</b>, HRE-Luc assay in 1%pO₂-exposed ctrl/shBeta MCF7 cells; <b>C</b>, CA9-Luc and SNAI2-Luc assay in ctrl/Beta-wt transfected and in ctrl/shBeta MCF7 cells under Nor/1%pO₂ conditions; <b>D</b>, CA9 and SNAI2 mRNA stability assay following inhibition of Polymerase 2 transcriptional activity by actinomycin D (100ng/ml) in ctrl/shBeta MCF7 cells exposed to 1%pO₂; <b>E</b>, Schematic representation of the HIF1-alpha/beta-catenin interplay in breast cancer cells in response to hypoxia: HIF1-alpha promotes transcription and cytoplasmic beta-catenin enhances stabilization of SNAI2 and CA9 mRNAs; the negative effect of beta-catenin on HIF-1alpha-induced transcription is also depicted. Data are presented as mean +/- s.d.; p values refers to t test. n=3, unless otherwise specified.</p