Interferon/STAT1 and neuregulin signaling pathways are exploratory biomarkers of cetuximab (Erbitux�) efficacy in KRAS wild-type squamous carcinomas: A pathway-based analysis of whole human-genome microarray data from cetuximab-adapted tumor cell-line models

Abstract

Abstract. KRAS mutation status is being used as the sole biomarker to predict therapeutic efficacy of cetuximab in metastatic colorectal cancer (mCRC). A significant number of mCRC patients with KRAS wild-type (WT) tumors, however, do not benefit from cetuximab. We are also lacking efficacy predictors in head and neck squamous cell carcinomas with an intact KRAS signaling and in non-small cell lung cancer in which KRAS mutations do not predict cetuximab efficacy. We recently established pre-clinical models of EGFR gene-amplified KRAS WT A431 squamous carcinoma cells chronically adapted to grow in the presence of cetuximab. We employed the ingenuity pathway analysis software to functionally interpret data from Agilent's whole human genome arrays in the context of biological processes, networks, and pathways. Cetuximabinduced activation of the interferon (IFN)/STAT1 appeared to switch from 'growth inhibitory' in acutely-treated cells to 'prosurvival' in chronically-adapted cells. Cetuximab treatment appeared to negatively select initially dominant IFN-sensitive clones and promoted selection of IFN-and cetuximabrefractory tumor clones constitutively bearing an up-regulated IFN/STAT1 signaling. High-levels of mRNAs coding for the EGFR ligands amphiregulin (AREG), epiregulin (EREG), and neuregulin-1/heregulin (NRG1) predicted for acute cetuximab's functioning. Chronic cetuximab, however, appeared to negatively select initially dominant AREG/EREG/NRG1-positive clones to promote selection of cetuximab-refractory clones exhibiting a knocked-down neuregulin signaling. Our current evolutionary mapping of the transcriptomic changes that occur during cetuximab-induced chronic blockade of EGFR/KRAS WT signaling strongly suggests that mRNAs coding for IFN/ STAT1-and EGFR ligands-related genes can be evaluated as novel predictors of efficacy in KRAS WT squamous cancer patients being treated with cetuximab.

Epidermal growth factor receptor inhibitors trigger a type I interferon response in human skin

“…EGFR-dependent down-regulation of IRF1 and, as a consequence, of IRF1-dependent type III IFN-λ, was shown to be the strategy exploited by some respiratory viruses to suppress the antiviral response in human airway epithelial cells, a mechanism effectively reverted by EGFR inhibition [42]. Finally, very similar to what we found in keratinocytes, cetuximab induced type I IFN/STAT1 signature in A431 human epidermal carcinoma cell line harbouring oncogenic Ras, with enhanced transcription of STAT1, a number of antiviral effectors (IFIT1, IFITM1, RSAD2, OAS1, and OAS2 among others), and also pro-inflammatory mediators, including TNF-α and very high lymphotoxin beta [43]. From these and our own findings, we can reasonably hypothesize that sustained activity of the EGFR/Ras/Raf/MEK/ERK phosphorylation cascade might exert a permanent, tonic regulation on IRF1 functional state, leading to effective control on the transactivation of a subset of genes which includes IFN-κ.…”

STAT1-Induced HLA Class I Upregulation Enhances Immunogenicity and Clinical Response to Anti-EGFR mAb Cetuximab Therapy in HNC Patients

“…Indeed, we observed STAT-1, HLA class I upregulation in cetuximab-treated patients in a novel neoadjuvant trial, suggesting that EGFR inhibition and/or IFNγ release contributes to the reversal of HLA downregulation. Results from recent studies also indicated an immunosuppressive effect of EGFR signaling on STAT1-dependent HLA class I and CIITA genes (38,39). However, HLA class I and CIITA are modulated by both total STAT1 and p-STAT1 (40,41), and a recent report also indicates that EGFR inhibitors reduces PD-L1 expression in lung tumors (42), suggesting multiple immune escape mechanisms mediated by EGFR signaling.…”

Cross-suppression of EGFR ligands amphiregulin and epiregulin and de-repression of FGFR3 signalling contribute to cetuximab resistance in wild-type KRAS tumour cells

“…Western blots were performed using an SDS–PAGE electrophoresis system as described previously (Oliveras-Ferraros et al , 2011a, 2011b), employing anti-AREG (Santa Cruz Biotechnology, Santa Cruz, CA, USA, cat. no.…”

“…Our recently described transcriptome signature for Ctx efficacy in cultured models of squamous cell carcinomas (SCCs) revealed that the molecular function of Ctx depended on high expression levels of genes encoding EGFR ligands (such as AREG and EREG ) concomitant with downregulation of DUSP -regulated EGFR inhibition (Oliveras-Ferraros et al , 2011a, 2011b). A dramatic downregulation of AREG/EREG mRNA expression, but not of other EGFR ligands, has been found to correlate with loss of Ctx efficacy (Oliveras-Ferraros et al , 2011a, 2011b).…”

“…Our recently described transcriptome signature for Ctx efficacy in cultured models of squamous cell carcinomas (SCCs) revealed that the molecular function of Ctx depended on high expression levels of genes encoding EGFR ligands (such as AREG and EREG ) concomitant with downregulation of DUSP -regulated EGFR inhibition (Oliveras-Ferraros et al , 2011a, 2011b). A dramatic downregulation of AREG/EREG mRNA expression, but not of other EGFR ligands, has been found to correlate with loss of Ctx efficacy (Oliveras-Ferraros et al , 2011a, 2011b). Therefore, we sought to establish the following: (a) whether cross-regulation mechanisms can explain the impact of AREG/EREG on Ctx efficacy; (b) whether the loss of AREG or EREG is sufficient to fully establish tumour resistance to Ctx; (c) whether the downregulation of AREG/EREG expression is indispensable for the Ctx mechanism of action; and/or (d) whether kinase-switching phenomena might contribute to bypass loss of EGFR-ligand signalling caused by Ctx.…”