Cetuximab-resistant Cell Research Articles

HOXB8 mediates resistance to cetuximab in colorectal cancer cells through activation of the STAT3 pathway

Homeobox B8 (HOXB8) belongs to the HOX family and was essential to the development of colorectal carcinoma. Among the prevalent monoclonal antibodies for treating RAS/BRAF wild-type metastatic colorectal cancer (mCRC) patients, cetuximab stands out, but resistance to cetuximab frequently arises in targeted treatments. Currently, the role of HOXB8 in cetuximab-resistant mCRC remains unclear. By comparing drug-sensitive cell lines (SW48) with drug-resistant cell lines (HCT116, CACO2), we discovered that HOXB8 was substantially expressed in cetuximab-resistant cell lines, and furthermore, in drug-resistant cell lines (HCT116, CACO2), HOXB8 knockdown increased the cytotoxicity of cetuximab via blocking the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Conversely, the excessive expression of HOXB8 reduced the growth suppression in SW48 cells caused by cetuximab by triggering the STAT3 signaling pathway. Conclusively, we conclude that HOXB8 has played an essential role in cetuximab-resistant mCRC and that treating HOXB8 specifically may be a useful treatment approach for certain cetuximab-resistant mCRC patients.

CircHIF1A induces cetuximab resistance in colorectal cancer by promoting HIF1α-mediated glycometabolism alteration

Epidermal growth factor receptor (EGFR)-targeted therapy is an important treatment for RAS wild-type metastatic colorectal cancer (mCRC), but the resistance mechanism remains unclear. Here, the differential expression of circRNAs between Cetuximab sensitive and resistant cell lines was analyzed using whole-transcriptome sequencing. We identified that the expression of circHIF1A was significantly higher in LIM1215-R than in LIM1215. When treated with Cetuximab, downregulation of circHIF1A level weakened the proliferation and clonal formation ability of LIM1215-R, caused more cells to enter G0-G1 phase, and significantly reduced the basal respiration, ATP production, and maximal respiration, as well as the glycolytic capacity and glycolytic reserve. The response rate and prognosis of circHIF1A-positive patients were inferior to those of negative patients. Mechanistically, circHIF1A can upregulate the level of hypoxia-inducible factor 1 A (HIF1A) by competitively binding to miR-361-5p, inducing the overexpression of enzymes such as glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA). In a xenograft model, inhibition of circHIF1A expression increased the sensitivity to Cetuximab treatment. In conclusion, circHIF1A can promote HIF1α-mediated glycometabolism alteration to induce Cetuximab resistance in CRC. It has the potential to become a screening indicator for the Cetuximab beneficial population in mCRC and a new therapeutic target for enhancing treatment efficacy.

3-Bromopyruvate overcomes cetuximab resistance in human colorectal cancer cells by inducing autophagy-dependent ferroptosis.

Colorectal cancer (CRC) remains a leading cause of cancer-related death worldwide. Cetuximab, in combination with chemotherapy, is effective for treating patients with wild-type KRAS/BRAF metastatic CRC (mCRC). However, intrinsic or acquired drug resistance often limits the use of cetuximab. In this study, we investigated the potential of co-treatment with 3-Bromopyruvate (3-BP) and cetuximab to overcome cetuximab resistance in CRC, both in vitro and in vivo. Our results demonstrated that the co-treatment of 3-BP and cetuximab synergistically induced an antiproliferative effect in both CRC cell lines with intrinsic cetuximab resistance (DLD-1 (KRASG13D/-) and HT29 (BRAFV600E)) and in a cetuximab-resistant cell line derived from Caco-2 with acquired resistance (Caco-2-CR). Further analysis revealed that co-treatment induced ferroptosis, autophagy, and apoptosis. Mechanistically, co-treatment inhibited FOXO3a phosphorylation and degradation and activated the FOXO3a/AMPKα/pBeclin1 and FOXO3a/PUMA pathways, leading to the promotion of ferroptosis, autophagy, and apoptosis in DLD-1 (KRASG13D/-), HT29 (BRAFV600E), and Caco-2-CR cells. In conclusion, our findings suggest that co-treatment with 3-BP and cetuximab could be a promising strategy to overcome cetuximab resistance in human CRC.

Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines.

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.

EGFR targeting of [177Lu] gold nanoparticles to colorectal and breast tumour cells: Affinity, duration of binding and growth inhibition of Cetuximab-resistant cells

ObjectiveRadioimmunotherapy (RIT) is a systemic therapy currently used in the treatment of patients with lymphoma. RIT complexes consist of a targeting molecule, commonly an antibody, radionuclide chelates and a linker which can be a nanoparticle platform. Nanoparticles facilitate the attachment of multiple radionuclides and targeting groups to a single complex. Here the target affinity, duration of target association and inhibition of colony formation of Cetuximab-resistant tumour cells with Cetuximab-targeted [177Lu]-AuNPs were investigated. Dose distribution in xenografts derived from EGFR-overexpressing cells was also determined. MethodsCetuximab-targeted [177Lu]-AuNPs were generated by functionalising 15nm AuNPs with the chelator DOTA and Cetuximab and radiolabelling with 177LuCl3. KDis, a measure of affinity, was determined by competitive binding to EGFR expressing cells. Radio-sensitivity was determined in EGFR expressing tumour cells including the Cetuximab resistant cell line HCT116 using a colony formation assay. Dose distribution was measured in sections from xenografts grown in nude mice using autoradiography. ResultsKDis for the complex binding to EGFR on MDA-MB-468 cells was 20 nM. Loss of cell associated [177Lu] activity was biphasic with loss of about 50% of activity in about 4 h. Remaining activity dissociated over a period of about 4 days. HCT8 and MDA-MB-468, but not HCT116 cells were sensitive to the growth inhibitory effect of Cetuximab. However, treatment with Cetuximab-targeted [177Lu]-AuNPs inhibited colony formation in all 3 cell lines. Dose distribution across sections from xenografts was found to demonstrate a co-efficient of variation of 15%. ConclusionCetuximab-targeted [177Lu]-AuNPs demonstrate high affinity for EGFR and could be an effective treatment for Cetuximab-resistant colorectal cancer cells. A strategy involving pre-treatment with receptor targeted[177Lu] to improve RIT therapeutic ratios has the potential to enhance clinical outcomes.

Abstract 1095: Overcoming acquired cetuximab resistance in head and neck squamous cell carcinoma: An in vitro study on the potential of PI3K inhibition

Abstract Resistance to therapies targeting the epidermal growth factor receptor (EGFR), such as cetuximab, remains a major roadblock in the search for effective therapeutic strategies in head and neck squamous cell carcinoma (HNSCC). Increasing evidence suggests that aberrant signalling of the PI3K/Akt pathway is involved in resistance to cetuximab treatment. Therefore, this study aims to investigate whether combined inhibition of EGFR and PI3K might be a novel therapeutic strategy to overcome acquired cetuximab resistance in HNSCC. Acquired cetuximab resistant HNSCC cell lines (SC263-R and SCC22b-R) were generated by chronically exposing initially sensitive cell lines to cetuximab. In parallel, control cell lines (SC263-PBS and SCC22b-PBS) were established by exposing these cells to the vehicle control (PBS). All cell lines were HPV-negative. Cytotoxicity of cetuximab (72h, 25 and 50 nM) plus the PI3K inhibitor buparlisib (72h, 0-1μM) was assessed using the Tecan Spark Cyto. Cells were stained with Hoechst 33342 and Cytotox Green (Sartorius) after 72h of treatment. Possible synergism between cetuximab and buparlisib was determined by calculating the combination index (CI) using the additive model. CI<0.8, CI = 1.0±0.2, and CI>1.2 indicated synergism, additivity, and antagonism, respectively. The effects of the combination treatment on cell cycle arrest and apoptosis were analysed flow cytometrically using the Vindelov and Annexin V-FITC/PI method, respectively. Simultaneous exposure to cetuximab and buparlisib revealed a synergistic interaction in the SC263-PBS and SC263-R cell lines (CI≤0.797). Furthermore, the percentage of cell death was the highest in the combination treatments, with a maximum of 30.33±4.18% for 50 nM cetuximab plus 1 μM buparlisib. Cetuximab monotherapy induced a small cell cycle arrest, while buparlisib monotherapy had little to no effect on cell cycle distribution. The addition of buparlisib to cetuximab resulted in a small increase in G0/G1 phase cells. Therapy-induced apoptosis was limited when cells were treated with cetuximab monotherapy. Buparlisib monotherapy induced a small percentage of apoptosis in a concentration-dependent manner. The addition of buparlisib to cetuximab resulted in an increase in the percentage of apoptotic cells with a maximum of 22.30±7.48%. These results are currently being validated in other acquired resistant cell lines. Preliminary results also show an additive to synergistic interaction between both drugs in the SCC22b-PBS and SCC22b-R cell lines. In conclusion, these results can serve as a preclinical rationale for a novel therapeutic strategy combining buparlisib with cetuximab in HNSCC patients who experience acquired cetuximab resistance. Further research is necessary to obtain a more profound understanding of the molecular mechanisms underlying this targeted combination therapy. Citation Format: Hannah Zaryouh, Hasan Baysal, Patrick Pauwels, Marc Peeters, Jan B. Vermorken, Filip Lardon, An Wouters, Ines De Pauw. Overcoming acquired cetuximab resistance in head and neck squamous cell carcinoma: An in vitro study on the potential of PI3K inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1095.

NMR Profiling of Ononis diffusa Identifies Cytotoxic Compounds against Cetuximab-Resistant Colon Cancer Cell Lines.

In the search of new natural products to be explored as possible anticancer drugs, two plant species, namely Ononis diffusa and Ononis variegata, were screened against colorectal cancer cell lines. The cytotoxic activity of the crude extracts was tested on a panel of colon cancer cell models including cetuximab-sensitive (Caco-2, GEO, SW48), intrinsic (HT-29 and HCT-116), and acquired (GEO-CR, SW48-CR) cetuximab-resistant cell lines. Ononis diffusa showed remarkable cytotoxic activity, especially on the cetuximab-resistant cell lines. The active extract composition was determined by NMR analysis. Given its complexity, a partial purification was then carried out. The fractions obtained were again tested for their biological activity and their metabolite content was determined by 1D and 2D NMR analysis. The study led to the identification of a fraction enriched in oxylipins that showed a 92% growth inhibition of the HT-29 cell line at a concentration of 50 µg/mL.

Identification of transcriptomic determinants to predict the efficacy of anti-EGFR treatment of colorectal cancer patients.

130 Background: It is now well known that in colorectal cancer (CRC) activating mutations in KRAS, NRAS, and BRAF confer resistance to anti-EGFR therapy. However, only ~ 40% of patients with RASWT (KRASWT/NRASWT/BRAFWT) tumors respond to therapy. We have used transcriptomic data in addition to somatic mutations to decipher intrinsic resistance mechanisms and identify novel biomarkers of anti-EGFR response in CRC. Methods: The transcriptomic profiles of metastatic CRC patients treated with the anti-EGFR drug cetuximab plus chemotherapy (n = 193) or as a single agent (n = 80) were retrospectively obtained from two independent clinical cohorts (Okita et al., 2018; & Khambata-Ford et al., 2007). Tumor samples were further divided into RASWT and RASMut groups and subsequently in four consensus molecular subtypes (CMS) based on their transcriptomic profile. The transcriptomic profiles of cetuximab treated CRC cell lines (n = 148, Medico et al., 2015) and PDX models (n = 230, Bertotti et al., 2015) were used for validation. Results: In RASWT population of cetuximab plus chemotherapy cohort, the disease control rates (DCR) observed for cetuximab in the second line were 92% (33/36) for CMS2 and 83% (15/18) for CMS4 tumors relative to 50% (2/4) and 59% (13/22) for CMS1 and CMS3 respectively (Chi-square test p-value = 0.037). Similarly, in cetuximab single agent cohort, the DCR were 0% (0/4), 68% (15/22), and 29% (5/17) for CMS1, CMS2, and CMS4, respectively (Chi-square test p-value = 0.029). In the preclinical study of 148 CRC cell lines, 60% (12/20) of CMS2 cell lines with RASWT genes were sensitive to cetuximab, versus no sensitive cell lines from other CMS (CMS1 = 0/4; CMS3 = 0/5; CMS4 = 0/12). In the PDX study, 84% (11/13) of CMS2 tumor showed clinical benefit, followed by CMS4 with 75% (6/8) in RASWT group (CMS1 = 2/5; CMS3 = 2/4). Gene Set Enrichment Analysis (GSEA) of tumor tissue samples identified that Myc (NES = 1.32, FDR = 0.3), E2F (NES = 2.24, FDR = 0.02), and mTOR (NES = 1.54, FDR = 0.1) pathways were active in the cetuximab refractory RASWT CMS2 patients relative to cetuximab sensitive tumors. Similarly, these pathways were also activated (FDR ≤ 0.05) in cetuximab resistant RASWT cell lines and PDX models, suggesting that preclinical models are appropriate for testing of potential therapeutic combinations to overcome resistance to anti-EGFR therapy. Conclusions: Resistance to anti-EGFR therapy in extended RASWT CRC tumors is mediated in part by transcriptional activation and can be predicted by consensus molecular subtype (CMS) and quantifying transcriptional states of Myc, E2F, and mTOR pathway gene sets.

GLUD1 Promotes Cetuximab Resistance in Metastasis Colorectal Cancer

Background: Colorectal cancer (CRC) is one of the most common causes of cancer mortality worldwide, among which metastatic colorectal cancer (mCRC) is the deadliest one. Cetuximab as a monoclonal antibody targeting epidermal growth factor receptor (EGFR) has improved the survival of mCRC patients. However, primary and acquired resistance to cetuximab are often found during therapy. Therefore, it is important to study the mechanism of cetuximab resistance and find more potential biomarkers. Methods: We previously established the acquired cetuximab resistant cell lines Caco2-CR and NCI-H508-CR by treating their parental cetuximab sensitive colorectal cancer cell lines, Caco2 and NCI-H508, with continuously increasing concentrations of cetuximab. Microarray analysis was utilized to detect the expression profiling of resistant cells and sensitive cells. Gene expression data of mCRC treated with cetuximab (GSE5851) was obtained from GEO. Patient-derived xenografts (PDX) were established using the tumor tissues from either primary lesions or liver metastases of mCRC patients without previous treatment. The metabolic flux of 13 C labelled glutamine in NCI-H508-CR and its parental cells was analyzed. Findings: Microarray analysis indicated that GLUD1, a key enzyme in glutamine metabolism, was one of the differentially expressed genes between Caco2-CR and Caco2. The expression of GLUD1 in the cetuximab resistant patients from GSE5851 dataset were higher than that in the sensitive ones. The same results were seen in animal PDX models. The glutamine metabolism was stronger in NCI-H508-CR compared with NCI-H508 and inhibition of glutamine metabolism could reduce the cetuximab resistance. GLUD1 could promote cetuximab resistance by generating more α-KG to support tricarboxylic acid (TCA) cycle. Furthermore, the expression of GLUD1 in resistant cells could be promoted by the nucleus translocated FOXO3. Interpretation: In this study，we found that GLUD1 can promote cetuximab resistance in mCRC, and it was transcriptional regulated by FOXO3. Funding: This work was supported by the National Natural Science Foundation of China (81772511). Declaration of Interest: None to declare. Ethical Approval: This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University

Abstract 3371: Role of the epidermal growth factor receptor expression and internalization in cetuximab-induced antibody-dependent cellular cytotoxicity in head and neck squamous cell carcinoma

Abstract The epidermal growth factor receptor (EGFR)-targeted therapies and immunotherapies are now at the forefront for treatment of head and neck squamous cell carcinoma (HNSCC). However, resistance to cetuximab, an anti-EGFR monoclonal antibody (mAb), is a major clinical problem, limiting the clinical benefit of treatment. Besides inhibition of downstream signalling, cetuximab can also induce antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to investigate the role of baseline EGFR expression and EGFR internalization following treatment with cetuximab as possible mechanisms that may explain differences in cetuximab-induced ADCC between HNSCC cell lines. Ten HNSCC cell lines, comprising cetuximab sensitive as well as intrinsically and acquired resistant cell lines, differing in HPV-status, were used. ADCC was assessed using the xCELLigence RTCA system, by co-culturing HNSCC cells with natural killer cells from healthy volunteers (E:T; 5:1). Baseline EGFR expression was flow cytometrically determined, following staining with an EGFR PE-conjugated antibody or a PE-conjugated isotype control. Experiments were performed under normoxic and hypoxic (1% O2) conditions. To determine EGFR internalization, cetuximab and the corresponding isotype control were stained with FabFluor-pH reagent prior to incubation with HNSCC cells. Phase-contrast and red fluorescence kinetics were quantified using the IncuCyte Zoom. We demonstrate that cetuximab-induced ADCC was highest in intrinsically cetuximab resistant cell lines compared to the acquired resistant and cetuximab sensitive cell lines. Under hypoxic conditions, a reduced ADCC was observed in the cetuximab sensitive and acquired resistant HNSCC cell lines but not in the intrinsically resistant cell lines. Baseline EGFR was expressed in a cell line-dependent manner and was not correlated with cetuximab sensitivity or HPV-status. Moreover, there was no correlation between baseline EGFR expression and the amount of cetuximab-induced ADCC. Importantly, the intrinsically cetuximab resistant cells demonstrated the highest level of EGFR internalization compared to the acquired resistant and cetuximab sensitive cell lines. Correlation analysis showed that the degree of EGFR internalization positively corelated with cetuximab-induced ADCC. In conclusion, we showed that induction of ADCC is maintained, independent of EGFR expression. Furthermore, internalization of EGFR might play a role in cetuximab resistance and ADCC-induction. Future research further unravelling this mechanism will provide a better understanding of mAb-based anti-EGFR therapies in the future. Citation Format: Hasan Baysal, Ines De Pauw, Hannah Zaryouh, Marc Peeters, Jan Baptist Vermorken, Evelien Smits, Filip Lardon, Julie Jacobs, An Wouters. Role of the epidermal growth factor receptor expression and internalization in cetuximab-induced antibody-dependent cellular cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3371.

Cetuximab-induced natural killer cell cytotoxicity in head and neck squamous cell carcinoma cell lines: investigation of the role of cetuximab sensitivity and HPV status

BackgroundThe epidermal growth factor receptor (EGFR) is overexpressed by 80–90% of squamous cell carcinoma of head and neck (HNSCC). In addition to inhibiting EGFR signal transduction, cetuximab, a monoclonal antibody targeting EGFR can also bind to fragment crystallisable domain of immunoglobulins G1 present on natural killer (NK), causing antibody-dependent cellular cytotoxicity (ADCC). However, presence of cetuximab resistance limits effective clinical management of HNSCC.MethodsIn this study, differences in induction of ADCC were investigated in a panel of ten HNSCC cell lines. Tumour cells were co-cultured with NK cells and monitored using the xCELLigence RTCA.ResultsWhile ADCC was not influenced by HPV status, hypoxia and cetuximab resistance did affect ADCC differentially. Intrinsic cetuximab-resistant cell lines showed an increased ADCC induction, whereas exposure to hypoxia reduced ADCC. Baseline EGFR expression was not correlated with ADCC. In contrast, EGFR internalisation following cetuximab treatment was positively correlated with ADCC.ConclusionThese findings support the possibility that resistance against cetuximab can be overcome by NK cell-based immune reactions. As such, it provides an incentive to combine cetuximab with immunotherapeutic approaches, thereby possibly enhancing the anti-tumoural immune responses and achieving greater clinical effectiveness of EGFR-targeting agents.

In vitro measurement of glucose uptake after radiation and cetuximab treatment in head and neck cancer cell lines using 18F-FDG, gamma spectrometry and PET/CT.

The standard treatment for head and neck squamous cell carcinoma (HNSCC) is radiotherapy, often in combination with chemotherapy or surgery. However, a novel monoclonal antibody, cetuximab (Erbitux®), has also been approved for patient therapy. The aim of present study was to develop an in vitro method for the measurement of 18F-fluoro-2deoxy-D-glucose (FDG) to determine if cellular 18F-FDG uptake is associated with response to radiotherapy or cetuximab treatment. In the current study, HNSCC cell lines were treated with radiation or with cetuximab. Next, the uptake of 18F-FDG was measured using a gamma spectrometer (GS). Thereafter, uptake after radiation was measured first with GS and then compared with positron emission tomography (PET)/computed tomography (CT) imaging. Furthermore, the mRNA expression of glucose transporter 1 (GLUT1) was measured following cetuximab treatment via reverse transcription-quantitative PCR. A study protocol was developed to measure the cellular uptake of 18F-FDG via gamma-ray spectrometry and comparable results were obtained with those of clinical PET/CT. The results revealed a decrease in 18F-FDG after radiation and cetuximab treatment. The uptake of 18F-FDG following cetuximab treatment was significantly lower in the cetuximab-sensitive cell line UT-SCC-14 compared with the cetuximab-resistant cell lines UT-SCC-2 and UT-SCC-45. Furthermore, after treatment with cetuximab for 24 and 48 h, a significant increase in GLUT1 expression was detected in the sensitive cell line compared with the two resistant cell lines. In conclusion, a novel yet reliable method for the measurement of intracellular 18F-FDG via GS has been developed, and our results indicate that 18F-FDG uptake is associated with radiation and cetuximab response in HNSCC.

Differential escape mechanisms in cetuximab-resistant head and neck cancer cells

Acquired cetuximab resistance is a challenge for oncologists treating advanced head and neck carcinoma (HNC). While intrinsic cetuximab resistance mechanism in colorectal cancer is known, resistance in HNC is unclear. We established two different cetuximab resistant HNC cell lines by culturing epidermal growth factor (EGFR) expressing UM–SCC–1 and UM–SCC–6 cell lines in the presence of 5 μg/ml cetuximab. We then explored potential mechanisms of resistance. We found that the 2 cell lines developed resistance by different mechanisms. Specifically, we found that UM–SCC–1 resistant cells (UM–SCC–1R) showed enhanced EGF-induced downstream signals while UM–SCC–6 resistant cells (UM–SCC–6R) demonstrated EGF-independent signaling. Global kinase activity (kinomic) profiling revealed unique signaling differences in the two resistant cell lines. However, both of the resistant lines demonstrated increased phospho-serine 727 and total STAT3 expression compared to the parental lines. STAT3 knockdown promoted increased cytotoxicity both in the presence and absence of cetuximab in the resistant lines suggesting that STAT3 may be a common target in cetuximab resistance.

ErbB4/KITENIN-Mediated Signaling is Activated in Cetuximab-Resistant Colorectal Cancer Cells.

ErbB4/KITENIN signaling plays a role in epidermal growth factor receptor (EGFR)-independent EGF pathways mediating the invasiveness and tumorigenesis of colorectal cancer cells. However, whether alterations in ErbB4/KITENIN signaling play a role in the resistance to anti-EGFR therapy remains unclear. Here, we established cetuximab-resistant DLD1 and HT29 cells, and analyzed changes in ErbB4/KITENIN signaling. c-Jun, a final effector in ErbB4/KITENIN-mediated signaling, was upregulated, whereas KITENIN levels remained constant in both cetuximab-resistant cell lines. The phosphorylation of EGFR and ErbB4 was increased in cetuximab-resistant cells, suggesting that ErbB4/KITENIN signaling contributed to the acquisition of cetuximab resistance in the cells. Silencing of KITENIN and/or ErbB4 increased cetuximab sensitivity in cetuximab-resistant cells. This study is the first to report the activation of ErbB4/KITENIN-mediated signaling in cetuximab-resistant colorectal cancer cells and the potential clinical application of ErbB4/KITENIN-targeting therapy for overcoming anti-EGFR resistance.

Predictive role of UCA1-containing exosomes in cetuximab-resistant colorectal cancer

BackgroundPrimary or acquired resistance to cetuximab often occurs during targeted therapy in metastatic colorectal cancer (mCRC) patients. In many cancers, the key role of the long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) in anticancer drug resistance has been confirmed. Emerging evidence has shown that specific exosomal lncRNAs may serve as meaningful biomarkers. In this study, we hypothesize that exosomal UCA1 might predict the response to cetuximab in CRC patients.MethodsFirst, acquired cetuximab-resistant cell lines were generated, and UCA1 expressions in these cells and their exosomes were compared. We also systematically evaluate the stability of exosomal UCA1. Thereafter, the predictive value of exosomal UCA1 in CRC patients treated with cetuximab was evaluated. Finally, through cell apoptosis assays and immunofluorescence staining, we analyzed the role of UCA1-containing exosomes in conferring cetuximab resistance.ResultsUCA1 expression was markedly higher in cetuximab-resistant cancer cells and their exosomes. Exosomal UCA1 was shown to be detectable and stable in serum from CRC patients. In addition, circulating UCA1-containing exosomes could predict the clinical outcome of cetuximab therapy in CRC patients, and UCA1 expression was considerably higher in the progressive disease/stable disease patients than in the partial response/complete response patients. Furthermore, exosomes derived from cetuximab-resistant cells could alter UCA1 expression and transmit cetuximab resistance to sensitive cells.ConclusionsWe discovered a novel role of UCA1-containing exosomes, showed their capability to transmit drug resistance and investigated their potential clinical use in predicting cetuximab resistance.

Abstract LB-090: Activation of KITENIN/ErbB4-mediated signaling contributes to acquisition of cetuximab resistance in colorectal cancer cells

Abstract ErbB4/KITENIN signaling plays a role in epidermal growth factor receptor (EGFR)-independent EGF pathways mediating the invasiveness and tumorigenesis of colorectal cancer cells. However, whether alterations in ErbB4/KITENIN signaling play a role in the resistance to anti-EGFR therapy remains unclear. Here, we established cetuximab-resistant DLD1 and HT29 cells, and analyzed changes in ErbB4/KITENIN signaling. c-Jun, a final effector in ErbB4/KITENIN-mediated signaling, was upregulated, whereas KITENIN levels remained constant in both cetuximab-resistant cell lines. The phosphorylation of EGFR and ErbB4 was increased in cetuximab-resistant cells, suggesting that ErbB4/KITENIN signaling contributed to the acquisition of cetuximab resistance in the cells. Silencing of KITENIN and/or ErbB4 increased cetuximab sensitivity in cetuximab-resistant cells. This study is the first to report the activation of ErbB4/KITENIN-mediated signaling in cetuximab-resistant colorectal cancer cells and the potential clinical application of ErbB4/KITENIN-targeting therapy for overcoming anti-EGFR resistance. Citation Format: So-Yeon Park, Yi Yang, Rui Zhou, Woo Kyun Bae, Ik Joo Chung, Kyung Keun Kim, Hangun Kim. Activation of KITENIN/ErbB4-mediated signaling contributes to acquisition of cetuximab resistance in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-090.

Abstract 05: Untangling the gene-epigenome networks: Timing of epigenetic regulation of gene expression in acquired cetuximab resistance gene programs

Abstract The current study performs time course RNA-seq and DNA methylation profiling to determine the complex interactions between gene expression and epigenetic changes in acquired therapeutic resistance. The genomics era provides widespread characterization of the genomic landscape of tumors and has enabled precision treatment strategies. Currently, epidermal growth factor receptor (EGFR) inhibitors are the only FDA-approved targeted therapy for clinical use in head and neck squamous cell carcinoma (HNSCC). EGFR inhibitors are only effective in a subset of each of these tumors. Moreover, patients with de novo sensitivity to EGFR inhibitors often subsequently acquire resistance and succumb to their tumors. Numerous genetic and epigenetic alterations occur in tumors with acquired resistance. However, their timing and function remain unknown. Therefore, we develop new computational techniques to find gene programs associated with the acquisition of resistance. High-throughput transcriptional profiling enables unprecedented characterization of individual genes during cancer treatment. However, identifying and targeting mechanisms of EGFR resistance from these high-throughput data requires novel systems biology techniques that can discriminate altered cellular signaling pathways in response to cancer treatment. Therefore, we developed a new bioinformatics algorithm Coordinated Gene Activity in Pattern Sets (CoGAPS) to quantify multiplexed regulation and gene reuse in oncogenic signaling. To test this algorithm, we modified the HaCaT keratinocyte cell line model of premalignancy to simulate cancer cells with constitutive overexpression of wild-type EGFR and with activating mutations in HRAS and PIK3CA in a controlled genetic background. We apply CoGAPS to gene expression data from these models. This algorithm quantified relative changes in cellular signaling pathway activity in these data, not accessible to standard gene comparisons. Increases in CoGAPS pathway signatures from HRAS also occurred in gene expression data from the cetuximab resistant HNSCC cell line (1CC8) relative to its parental, sensitive cell line (UMSCC1). Investigation of the mechanisms of acquired resistance has previously been limited by reliance on case-control comparisons between sensitive and resistant cells, such as the UMSCC1 and 1CC8 cell lines. Since activity in cellular signaling pathways evolves during cetuximab resistance, it is essential to extend these case-control paradigms to quantify the dynamics responsible for resistance. Therefore, we developed a novel time course analysis to determine the molecular mechanisms of acquired cetuximab resistance in HNSCC. Specifically, we treated the cetuximab sensitive SCC25 HNSCC cell line over generations with both cetuximab and PBS. This long-term treatment protocol models the progression of acquired therapeutic resistance, including controls for clonal selection unrelated to treatment. Overexpression of the previous HRAS gene program also occurs in gene expression data measured during acquired cetuximab resistance in SCC25. We also measure DNA methylation during this time course to find the driver of this aberrant signaling associated with resistance. We apply CoGAPS analysis to the time course data for both DNA methylation and gene expression. This analysis distinguished early gene expression changes from cetuximab treatment from longer-term epigenetic alterations to gene expression during acquired resistance. Epigenetic regulation of FGFR1 expression emerged as the dominant mechanism of acquired therapeutic resistance in this system. Thus, our integration of time course DNA methylation and gene expression data enables unprecedented inference of the timing of targetable gene-epigenome programs responsible for acquired resistance. Citation Format: Genevieve Stein-O'Brien, Luciane T. Kagohara, Sijia Li, Manjusha Thakar, Ruchira Ranaweera, Michael Considine, Ludmila V. Danilova, Hiroyuki Ozawa, Joseph A. Califano, Daria A. Gaykalova, Michael F. Ochs, Christine H. Chung, Elana J. Fertig. Untangling the gene-epigenome networks: Timing of epigenetic regulation of gene expression in acquired cetuximab resistance gene programs [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 05.

Identification and validation of cetuximab resistance associated long noncoding RNA biomarkers in metastatic colorectal cancer

BackgroundCetuximab is one of the most widely used epidermal growth factor receptor (EGFR) inhibitors to treat patients with metastatic colorectal cancer (mCRC) harboring wild-type of RAS/RAF status. However, primary and acquired resistance to cetuximab is often found during target therapy. MethodsTo gain insights into the functions of long non-coding RNA (lncRNA) in cetuximab resistance, we used a lncRNA-mining approach to distinguish lncRNA specific probes in Affymetrix HG-U133A 2.0 arrays. Then we performed lncRNA expression profiling in a cetuximab treated mCRC cohort from Gene Expression Ominus (GEO). The potential lncRNAs were further validated in acquired cetuximab resistant cell lines and clinical samples of our hospital. The functions and associated pathways of the prognostic lncRNA were predicted by GO and KEGG analyses. Results249 lncRNA-specific probe sets (corresponding to 212 lncRNAs) were represented in Affymetrix HG-U133A 2.0 arrays. We found that 9 lncRNAs were differentially expressed between disease control group (DCG) and non-responders, and 5 of these 9 lncRNAs were significantly related with the progression-free survival (PFS) of the patients. Among those 5 lncRNAs, POU5F1P4 was also down-regulated in acquired cetuximab resistant cells, as well as in cetuximab resistant patients. Downregulation of POU5F1P4 decreased the sensitivity of colorectal cancer cells to cetuximab. ConclusionOur findings indicate the potential roles of lncRNAs in cetuximab resistance, and may provide the useful information for discovery of new biomarkers and therapeutic targets.

Abstract 4102: Overcoming intrinsic and acquired cetuximab resistance: an in vitro study on the potential of afatinib in HNSCC cell lines

Abstract Aberrant signalling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumorigenesis of many cancer types, including head and neck squamous cell carcinoma (HNSCC), making it a compelling drug target. After the initial promising results of EGFR-targeted therapies, the problem of therapeutic resistance is emerging and new treatment options are necessary. In contrast to the first generation EGFR inhibitors, afatinib is an irreversible ErbB family blocker that inhibits EGFR as well as HER2 and HER4. The objective of this study was to investigate whether afatinib is able to overcome intrinsic and acquired cetuximab resistance in human papillomavirus (HPV) positive and negative HNSCC cell lines under normal and reduced oxygen conditions and to identify the molecular mechanisms underlying afatinib’s cytotoxic effect. Sensitivity to cetuximab treatment was investigated in a panel of HPV positive and negative HNSCC cell lines using the colorimetric sulforhodamine B (SRB) assay (168h, 0-2μM). Cetuximab sensitive and intrinsically resistant cell lines were identified. Acquired cetuximab resistant cell lines were generated by chronically exposing initially sensitive cell lines to cetuximab. In parallel, control cell lines were established by exposing these cells to the vehicle control (PBS). Cytotoxicity of afatinib (72h, 0 - 5μM) was assessed under normoxic and hypoxic (1% O2) conditions. Cell cycle distribution and induction of apoptotic cell death were assessed flow cytometrically using the Vindelov method and Annexin V-FITC/PI assay, respectively. Data analysis was performed using WinNonlin and FlowJo software. We identified intrinsic cetuximab resistance in 5 out of 7 HNSCC cell lines (i.e. LICR-HN1, Cal-27, SQD9, 93-VU-147T and UM-SCC104) and generated acquired cetuximab resistant cell lines (i.e. SCC22b-R and SC263-R). Afatinib showed a clear concentration-dependent cytotoxic effect in both cetuximab sensitive and resistant cell lines with IC50 values ranging between 0.019 and 4.04 μM. Furthermore, afatinib maintained its cytotoxic effect under hypoxic conditions. Treatment with afatinib led to an increase of the proportion of cells in the G0/G1 phase of the cell cycle. Afatinib also induced an increase in the percentage of AnnV+/PI- and AnnV+/PI+ cells with a corresponding decrease of the percentage viable (AnnV-/PI-) cells. In conclusion, our results suggest that afatinib has the potential to overcome cetuximab resistance, as it was able to establish cytotoxicity in HPV positive and negative HNSCC cell lines that were intrinsically and acquired resistant to cetuximab. Furthermore, treatment with afatinib caused a G0/G1 cell cycle arrest and induced apoptotic cell death. These data support the hypothesis that afatinib might be a promising novel therapeutic strategy to treat HNSCC patients experiencing intrinsic or acquired cetuximab resistance. Citation Format: Ines De Pauw, An Wouters, Jolien Van den Bossche, Vanessa Deschoolmeester, Patrick Pauwels, Jan B. Vermorken, Marc Peeters, Filip Lardon. Overcoming intrinsic and acquired cetuximab resistance: an in vitro study on the potential of afatinib in HNSCC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4102. doi:10.1158/1538-7445.AM2017-4102

Abstract 5840: Radiosensitization with the c-MET inhibitor crizotinib in KRAS mutant colorectal cancer

Abstract Background: KRAS mutant colorectal cancers (CRC) are resistant to many targeted therapies including cetuximab. Novel molecular targets for KRAS mutant tumors may enhance the efficacy of chemoradiation. C-MET plays important roles in treatment resistance, tumor invasion, and metastasis. In this study, we used a small molecule inhibitor of c-MET, crizotinib in cetuximab resistant mutant KRAS driven CRC cell lines and assessed radiosensitization. Methods: To test the effect of c-MET inhibition on CRC cells we used the KRAS mutant cell lines HCT116, DLD1, and LoVo. Colony formation assays were performed to assess the effects of crizotinib and cetuximab. Immunoblot analysis was used to determine the effect of crizotinib on c-MET and downstream pathways including ERK and AKT phosphorylation. We then selected doses of crizotinib below the IC50 and assessed clonogenic survival with radiation. To study potential mechanisms of radiosensitization cell cycle analysis was performed using flow cytometry. The effect of c-MET inhibition on cellular function was assessed using cell migration assays. Results: We first performed a series of clonogenic survival assays with crizotinib and cetuximab to determine the IC50 for each cell line. As expected, the KRAS mutant cell lines LoVo, HCT116, and DLD1 were resistant to cetuximab (surviving fraction greater than 95% at 100 μg/ml). These cell lines were sensitive to the c-MET inhibitor crizotinib with IC50 values of 14.4 nM for LoVo, 316 nM for HCT166, and 217 nM for DLD1. Immunoblot analysis showed that crizotinib at concentrations near the IC50 for each cell line attenuated c-MET phosphorylation and downstream signaling pathways including ERK and AKT. Crizotinib further blocked radiation induced c-MET phosphorylation and attenuated activation of downstream signaling pathways. We then performed a series of clonogenic survival assays to study the radiosensitizing potential of crizotinib. Pretreatment with crizotinib for 24 hours radiosensitized LoVo and DLD1 cell lines with enhancement ratios of 1.66 and 1.25, respectively. Interestingly, treatment with crizotinib for 1 hour prior to radiation had only modest dose enhancement, suggesting that prolonged exposure to crizotinib is required for optimal radiosensitization. Cell cycle analysis revealed minimal G1 arrest with crizotinib. Additionally, crizotinib completely blocked HGF and EGF induced cell migration. Conclusions: Inhibition of c-MET with crizotinib effectively sensitizes cetuximab resistant KRAS mutant CRC cell lines to radiation. Given the important role c-MET plays in treatment resistance, crizotinib has the potential to improve the outcome of patients with locally advanced rectal cancer undergoing radiation therapy. Citation Format: Kyle C. Cuneo, Himabindu Kurapati, Ranjit Mehta, Theodore S. Lawrence, Mukesh K. Nyati. Radiosensitization with the c-MET inhibitor crizotinib in KRAS mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5840. doi:10.1158/1538-7445.AM2017-5840