Bernardo Queralt

Stem cell property epithelial‐to‐mesenchymal transition is a core transcriptional network for predicting cetuximab (Erbitux™) efficacy in KRAS wild‐type tumor cells

Beyond a well‐recognized effect of KRAS mutations in determining de novo inefficacy of cetuximab (CTX) in metastatic colorectal cancer, we urgently need a biomarker signature for predicting CTX efficacy in KRAS wild‐type (WT) tumors. CTX‐adapted EGFR gene‐amplified KRAS WT tumor cell populations were induced by stepwise‐chronic exposure of A431 epidermoid cancer cells to CTX. Genome‐wide analyses of 44K Agilents whole human arrays were bioinformatically evaluated by Gene Set Enrichment Analysis (GSEA)‐based screening of the KEGG pathway database. Molecular functioning of CTX was found to depend on: (i) The occurrence of a positive feedback loop on Epidermal Growth Factor Receptor (EGFR) activation driven by genes coding for EGFR ligands (e.g., amphiregulin); (ii) the lack of a negative feedback on mitogen‐activated protein kinase (MAPK) activation regulated by dual‐specificity phosphatases (e.g., DUSP6) and; (iii) the transcriptional status of gene pathways controlling the epithelial‐to‐mesenchymal transition (EMT) and its reversal (MET) program (actin cytoskeleton and cell–cell communication—e.g., keratins—focal adhesion signaling—e.g., integrins—and EMT‐inducing cytokines – e.g., transforming growth factor‐β). Quantitative real‐time PCR, high‐content immunostaining, and flow‐cytometry analyses confirmed that CTX efficacy depends on its ability to promote: (i) Stronger cell–cell contacts by up‐regulating the expression of the epithelial markers E‐cadherin and occludin; (ii) down‐regulation of the epithelial transcriptional repressors Zeb, Snail, and Slug accompanied by restoration of cortical F‐actin; and (iii) complete prevention of the CD44pos/CD24neg/low mesenchymal immunophenotype. The impact of EGFR ligands/MAPK phosphatases gene transcripts in predicting CTX efficacy in KRAS WT tumors may be tightly linked with the ability of CTX to concurrently reverse the EMT status, a pivotal property of migrating cancer stem cells. J. Cell. Biochem. 112: 10–29, 2011.

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

BACKGROUND:In addition to the mutational status of KRAS, the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG) might function as bona fide biomarkers of cetuximab (Ctx) sensitivity for most EGFR-driven carcinomas.METHODS:Lentivirus-delivered small hairpin RNAs were employed to specifically reduce AREG or EREG gene expression in wild-type KRAS A431 squamous cell carcinoma cells. Colony-forming assays were used to monitor the impact of AREG and EREG knockdown on Ctx efficacy. Amphiregulin and EREG protein expression levels were assessed by quantitative ELISA in parental A431 cells and in pooled populations of A431 cells adapted to grow in the presence of Ctx. A phosphoproteomic platform was used to measure the relative level of phosphorylation of 42 distinct receptor tyrosine kinases before and after the acquisition of resistance to Ctx.RESULTS:Stable gene silencing of either ligand was found to notably reduce the expression of the other ligand. Parental A431 cells with normal expression levels of AREG/EREG exhibited significantly increased growth inhibition in response to Ctx, compared with derivatives that are engineered to produce minimal AREG/EREG. The parental A431 cells acutely treated with Ctx exhibited reduced basal expression levels of AREG/EREG. Pooled populations of Ctx-resistant A431 cells expressed significantly lower levels of AREG/EREG and were insensitive to the downregulatory effects of Ctx. Phosphoproteomic screen identified a remarkable hyperactivation of FGFR3 in Ctx-resistant A431 cells, which gained sensitivity to the cytotoxic and apoptotic effects of the FGFR3 TK inhibitor PD173074. The A431 parental cells acutely treated with Ctx rapidly activated FGFR3 and their concomitant exposure to Ctx and PD173074 resulted in synergistic apoptosis.CONCLUSION:Cross-suppression of AREG/EREG expression may explain the tight co-expression of AREG and EREG, as well as their tendency to be more highly expressed than other EGFR ligands to determine Ctx efficacy. The positive selection for Ctx-resistant tumour cells exhibiting AREG/EREG cross-suppression may have an important role in the emergence of Ctx resistance. As de-repression of FGFR3 activity rapidly replaces the loss of EGFR-ligand signalling in terms of cell proliferation and survival, combinations of Ctx and FGFR3-targeted drugs may be a valuable strategy to enhance the efficacy of single Ctx while preventing or delaying acquired resistance to Ctx.British Journal of Cancer (2012) 106, 1406–1414. doi:10.1038/bjc.2012.103 www.bjcancer.com

Evolution of the predictive markers amphiregulin and epiregulin mRNAs during long-term cetuximab treatment of KRAS wild-type tumor cells

SummaryMolecular mechanisms other than activating KRAS mutations should underlie the occurrence of weaker versus stronger responses to cetuximab (CTX) in EGFR-dependent carcinomas with either an intact KRAS signaling or in which KRAS mutations do not predict CTX efficacy. We hypothesized that KRAS wild-type (WT) tumor cell-line models chronically adapted to grow in the presence of CTX could be interrogated to establish if the positive predictive value of the mRNAs coding for the EGFR ligands amphiregulin (AR) and epiregulin (EPI) could be significantly altered during and/or after treatment with CTX. Gene expression analyses using real-time (kinetic) RT-PCR were performed to monitor the transcriptional evolution of EGFR ligands EGF, TGFα, AR, BTC, EPI, NRG and HB-EGF in experimental modes induced to exhibit acquired resistance to the mono-HER1 inhibitor CTX, the mono-HER2 inhibitor trastuzumab (Tzb) or the dual HER1/HER2 inhibitor lapatinib (LPT). Gene expression signatures for EGFR ligands distinctively related to the occurrence of unresponsiveness to CTX, Tzb or LPT, with minimal overlap between them. CTX’s molecular functioning largely depended on the overproduction of the mRNAs coding for the EGFR ligands AR and EPI. Thus, a dramatic down-regulation of AR/EPI mRNA expression occurred upon loss of CTX efficacy in EGFR-positive tumor cells with an intact regulation of RAS signaling. Unlike KRAS mutations, which are informative of unresponsiveness to CTX solely in mCRC, our hypothesis-generating data suggest that expression status of AR and EPI mRNAs might be evaluated as dynamic predictors of response in KRAS WT patients receiving any CTX-based therapy.

Targeting STAT3 with silibinin to improve cancer therapeutics.

Signal transducer and activator of transcription 3 (STAT3) has a prominent role in mediating resistance to conventional chemo-/radio-therapies and modern targeted drugs. While a number of STAT3 inhibitors have been shown to enhance the efficacy of therapeutic agents in vitro, the majority of them have yet to enter clinical evaluation mostly because of lack of efficacy issues. Silibinin is the main component of the silymarin complex, a standardized extract obtained from the seeds of the milk thistle herb Silybum marianum. This review summarizes current evidence supporting the ability of silibinin to function as a natural down-modulator of STAT3 activity. We examine the reported capacity of silibinin to reduce the toxicity of cancer treatments and to reverse tumor cell resistance via STAT3 inhibition. We also briefly review our clinical data in cancer patients treated with oral nutraceutical products containing silibinin. The beneficial effects of silibinin might accelerate the design of strategies aimed to overcome and prevent the emergence of STAT3-mediated cancer drug resistance in clinical settings.

Transcriptional upregulation of HER2 expression in the absence of HER2 gene amplification results in cetuximab resistance that is reversed by trastuzumab treatment

The recent identification of HER2 gene amplification as a novel predictor of resistance to the EGFR (HER2)-targeted antibody cetuximab and of response to combination therapies against EGFR and HER2 in wild-type KRAS tumor settings may represent a further step toward personalized medicine for patients with colorectal cancer. Herein, we show that transcriptional upregulation of HER2 expression in the absence of HER2 gene amplification is a molecular phenomenon that takes place in EGFR-dependent, wild-type KRAS squamous cell carcinoma (SCC) cells that acquire resistance to cetuximab. Since cetuximab activity against cetuximab-refractory SCC cells can be fully restored in the presence of the anti-HER2 monoclonal antibody trastuzumab, our findings suggest that, beyond HER2 gene amplification, we might need to redefine the threshold values for HER2 positivity to improve the accuracy of the selection of cetuximab-refractory patients with wild-type KRAS that may benefit from receiving a cetuximab/trastuzumab combination.

Synthetic lethal interaction of cetuximab with MEK1/2 inhibition in NRAS -mutant metastatic colorectal cancer

KRAS mutations are an established predictor of lack of response to EGFR-targeted therapies in patients with metastatic colorectal cancer (mCRC). However, little is known about the role of the rarer NRAS mutations as a mechanism of primary resistance to the anti-EGFR monoclonal antibody cetuximab in wild-type KRAS mCRC. Using isogenic mCRC cells with a heterozygous knock-in of the NRAS activating mutation Q61K, we aimed to elucidate the mechanism(s) by which mutant NRAS blocks cetuximab from inhibiting mCRC growth. NRASQ61K/+ cells were refractory to cetuximab-induced growth inhibition. Pathway-oriented proteome profiling revealed that cetuximab-unresponsive ERK1/2 phosphorylation was the sole biomarker distinguishing cetuximab-refractory NRASQ61K/+ from cetuximab-sensitive NRAS+/+ cells. We therefore employed four representative MEK1/2 inhibitors (binimetinib, trametinib, selumetinib, and pimasertib) to evaluate the therapeutic value of MEK/ERK signaling in cetuximab-refractory NRAS mutation-induced mCRC. Co-treatment with an ineffective dose of cetuximab augmented, up to more than 1,300-fold, the cytotoxic effects of pimasertib against NRASQ61K/+ cells. Simultaneous combination of MEK1/2 inhibitors with cetuximab resulted in extremely high and dose-dependent synthetic lethal effects, which were executed, at least in part, by exacerbated apoptotic cell death. Dynamic monitoring of real-time cell growth rates confirmed that cetuximab synergistically sensitized NRASQ61K/+ cellsto MEK1/2 inhibition. Our discovery of a synthetic lethal interaction of cetuximab in combination with MEK1/2 inhibition for the NRAS mutant subgroup of mCRC underscores the importance of therapeutic intervention both in the MEK-ERK and EGFR pathways to achieve maximal therapeutic efficacy against NRAS-mutant mCRC tumors.

EphA2 receptor activation with ephrin-A1 ligand restores cetuximab efficacy in NRAS-mutant colorectal cancer cells

Patients with wild-type KRAS metastatic colorectal cancer (mCRC) that harbors NRAS activating mutations do not benefit from anti-EGFR therapies. Very little is known about oncogenic NRAS signaling driving mCRC unresponsiveness to the EGFR-directed antibody cetuximab. Using a system of paired NRAS-mutant and wild-type isogenic mCRC cell lines to explore signaling pathways engaged by the common oncogenic NRAS Q61K variant upon challenge with cetuximab, we uncovered an unexpected mechanism of resistance to cetuximab involving dysregulation of the ephrin-A1/EphA2 signaling axis. Parental NRAS+/+ cells, but not NRASQ61K/+ cells, activated the ephrin receptor ephA1 in response to cetuximab treatment. Moreover, whereas cetuximab treatment significantly downregulated EPHA2 gene expression in NRAS+/+ cells, EPHA2 expression in NRASQ61K/+ cells was refractory to cetuximab. Remarkably, pharmacologically mimicked ephrin-A1 engagement to ephA2 converted NRAS-mutant into RAS wild-type mCRC cells in terms of cetuximab efficacy. Accordingly, activation of the ephA2 receptor by bioactive recombinant human ephrin-A1/Fc-fusion protein suppressed the cetuximab-unresponsive hyperactivation of MAPK and AKT and fully restored cetuximab activity in NRAS-mutant colorectal cells. Collectively, these findings reveal that the clinical benefit of cetuximab in mCRC might necessarily involve the suppression of the ligandless oncogenic signaling of the ephA2 receptor. Hence, ligand-dependent tumor suppressor signaling using therapeutic ephA2 agonists might offer new therapeutic opportunities to clinically widen the use of cetuximab in NRAS-mutated and/or ephA2-dependent mCRC tumors.

Lymphopenia and changes on peripheral blood immunophenotype in patients with hepatobiliarypancreatic tumors treated with chemoradiotherapy.

e15610Background: Chemoradiation-related lymphopenia has been linked to prognosis in retrospective studies. The aim of this trial was to prospectively assess the clinical significance of lymphopeni...