Molecular mechanisms driving aberrant activation of EGFR pathway: implications for cancer treatment

Epidermal Growth Factor Receptor (EGFR) activates a robust signaling network to which tumors often become addicted. Over the last three decades, EGFR targeting has been intensely pursued as a treatment strategy for metastatic colorectal cancer (mCRC). One approach uses monoclonal antibodies (mAbs) to inhibit the extracellular domain of EGFR, thus blocking natural ligands binding. Unfortunately, patients often develop resistance, with consequent tumor growth and relapse. Tumors heterogeneity has been addressed as the main culprit for multiple escaping mechanisms, reflecting the high level of molecular heterogeneity in each metastatic site. Mechanisms of cancer cell resistance include either acquisition of new mutations or non-genomic activation of alternative signaling routes and, in this context, a role of IL-1 is emerging. For example IL-1 expression proved to be elevated in human breast, colon, lung and head and neck cancers, and patients with IL-1 producing tumors have generally bad prognosis. Our studies, performed with a cohort of 150 colorectal cancer xenopatients, associate poor response to CTX with increased abundance of a set of inflammatory cytokines, including IL-1A, IL-1B and IL-8. Stemming from these observations, our working hypothesis assumes that resistance to CTX is acquired, in a subset of CRC patients, through cell plasticity and consequent rewiring of signalling networks, which confer dependency on the IL1 pathway. This hypothesis foresees an auto-stimulatory feedback loop dependent on the IL-1 produced by the tumors, with consequent immunosuppression and tumor progression. Thus, combining IL-1 and EGFR neutralization may synergistically impair CRC response to CTX in vivo

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

A module of inflammatory cytokines defines resistance of colorectal cancer to EGFR inhibitors

Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A, IL1B and IL8, which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro. This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting

Aberrant MET activation impairs perinuclear actin cap organization with YAP1 cytosolic relocation

: Little is known about the signaling network responsible for the organization of the perinuclear actin cap, a recently identified structure holding unique roles in the regulation of nuclear shape and cell directionality. In cancer cells expressing a constitutively active MET, we show a rearrangement of the actin cap filaments, which crash into perinuclear patches associated with spherical nuclei, meandering cell motility and inactivation of the mechano-transducer YAP1. MET ablation is sufficient to reactivate YAP1 and restore the cap, leading to enhanced directionality and flattened nuclei. Consistently, the introduction of a hyperactive MET in normal epithelial cells, enhances nuclear height and alters the cap organization, as also confirmed by TEM analysis. Finally, the constitutively active YAP1 mutant YAP5SA is able to overcome the effects of oncogenic MET. Overall, our work describes a signaling axis empowering MET-mediated YAP1 dampening and actin cap misalignment, with implications for nuclear shape and cell motility

The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer

In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs)

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 interleukin 1 axis in the resistance to EGFR targeting antibody

Background Cetuximab (CTX) is a monoclonal antibody targeting the Epidermal Growth Factor Receptor (EGFR), which is commonly utilized to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, clinicians often observe a residual disease, with a population of cells surviving the treatment and eventually enabling CTX resistance. Our previous studies, performed with a cohort of 150 CRC xenopatients, associated poor response to CTX with increased abundance of a set of pro-inflammatory cytokines, including IL-1A, IL-1B and IL-8. IL-1 has been reported in the promotion of Senescence Associated Secretory Phenotype (SASP). Stemming from these observations, our current work aims to assess the effect of IL-1 pathway in CTX mediated senescence and resistance. Methods We employed a recombinant decoy (IL-1R1 TRAP), namely a soluble protein combining the human immunoglobulin Fc portion linked to the extracellular region of IL1-receptor, with the ability to sequester IL1 directly from the medium. We generated stable clones expressing a functional IL-1R1 TRAP, and tested the effects on proliferation of cancer cells growing both as monolayer and as 3D colonospheres. Next, the action of IL-1R1 TRAP decoy on EGFR downstream signalling pathways has been evaluated by analyses of MAPK and AKT axes. Results The analysis of colorectal cancer patients' datasets revealed that high levels of IL-1 receptor correlate with poor survival, specifically for the consensus molecular subtype 1 (CMS1). Next, mCRC patients not responding to CTX blockage, display higher level of IL-1R1 compared to the responsive ones. Furthermore, we found that IL-1R1 inhibition leads to decreased proliferation of colorectal cancer cells as consequence of a dampened MAPK and AKT signalling activation. Mechanistically, we show that CTX leads to a post-senescent phenotype as detected by bgalactosidase, HP1-g and p21 markers and stemness traits. Conclusions We are currently working on dissecting the contribution of IL1 signalling in resistance to CTX, as a consequence of the post-senescent phenotype. We conclude that IL1 signalling inhibition may represent a new therapeutic strategy suitable for patients who acquired refractoriness to EGFR targeted antibody therapy

Molecular mechanisms of resistance to cetuximab mediated by IL-1 signaling

Cetuximab (CTX) is a monoclonal antibody targeting Epidermal Growth Factor Receptor (EGFR), which is commonly employed to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, clinicians often observe a failure of the therapy with a population of cells surviving the treatment and eventually enabling CTX resistance. Our previous studies, employing a cohort of 150 CRC xenopatients, associated poor response to CTX with increase abundance of a set of inflammatory cytokines, namely IL-1, IL-1 and IL-8 (Gelfo et al., 2016). In the time frame of my first year of Ph.D, we found that in patients, undergoing CTX treatment, overexpression of IL-1 Receptor (IL-1R) correlates with reduced response. Stemming from these observations, we assumed that resistance to CTX is acquired, in a subset of CRC patients, through cell plasticity, as a consequent rewiring of signaling networks. Employing a recombinant decoy (IL1R-Fc) able to sequester IL-1 directly from the medium, our results show that IL1R decoy successfully dampens pSTAT3 activation along with MAPK and PI3K axes, thus decreasing proliferation and colon spheres formation. Furthermore, we report that IL1R abundance predicts disease relapse free survival in a cohort of 1700 colorectal cancer patients, and it appears associated to a specific subtype, namely the consensus molecular subtype 1 (CMS1). Our data therefore suggest that a loop mediated by the IL1 and its cognate receptor mediates CTX resistance in a specific subtype of colorectal cancer. Mechanistically, our preliminary data show that CTX treatment promotes an increase in inflammatory cytokines leading to a post-senescent phenotype, as detected by -galactosidase, HP1- and p21 markers

A putative role for interleukin 1 pathway in resistance to EGFR blockade

Cetuximab (CX) is a monoclonal antibody targeting the Epidermal Growth Factor Receptor (EGFR), which is commonly utilized to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, clinicians often observe a residual disease, with a population of cells surviving the treatment and eventually enabling CX resistance. Our previous studies, performed with a cohort of 150 CRC xenopatients, associated poor response to CX with increased abundance of a set of inflammatory cytokines, including IL1A, B and IL8. Stemming from these observations, our working hypothesis assumes that, resistance to CX is acquired, in a subset of CRC patients, through cell plasticity and consequent rewiring of signalling networks, which confer to tumors dependency on the IL1 pathway. In order to assess the effect of IL1 activity, we employed a colon cancer model unresponsive to cetuximab, as previously characterized in our laboratory. To inhibit activation of the IL1 pathway we used anakinra, an IL1-receptor antagonist and parthenolide, which modulates the activity of NF-kB, the transcription factor involved in the feed-forward loop of inflammation mediators. Furthermore, we employed a recombinant decoy (IL1R-Fc), namely a soluble protein combining the human immunoglobulin Fc portion linked to the extracellular region of IL1-receptor, with the ability to sequester IL1 directly from the medium. We generated stable clones of CXresistant cells expressing IL1R-Fc. Our preliminary results show that inhibition of IL1R leads to a proliferation decrease of colorectal cancer cells. These findings support the hypothesis of a compensatory activation of the IL1-receptor pathway in cetuximab-resistant CRC cells. Hence, modulating IL1 signalling might represent a new therapeutic strategy suitable for patients who acquired refractoriness to monoclonal antibody therapy