DiFi Cells Research Articles

Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer.

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80nm EV diameter for CytoFLEX and ∼10 PEs/∼67nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.

MCT1-dependent lactate recycling is a metabolic vulnerability in colorectal cancer cells upon acquired resistance to anti-EGFR targeted therapy

Despite the implementation of personalized medicine, patients with metastatic CRC (mCRC) still have a dismal overall survival due to the frequent occurrence of acquired resistance mechanisms thereby leading to clinical relapse. Understanding molecular mechanisms that support acquired resistance to anti-EGFR targeted therapy in mCRC is therefore clinically relevant and key to improving patient outcomes. Here, we observe distinct metabolic changes between cetuximab-resistant CRC cell populations, with in particular an increased glycolytic activity in KRAS-mutant cetuximab-resistant CRC cells (LIM1215 and OXCO2) but not in KRAS-amplified resistant DiFi cells. We show that cetuximab-resistant LIM1215 and OXCO2 cells have the capacity to recycle glycolysis-derived lactate to sustain their growth capacity. This is associated with an upregulation of the lactate importer MCT1 at both transcript and protein levels. Pharmacological inhibition of MCT1, with AR-C155858, reduces the uptake and oxidation of lactate and impairs growth capacity in cetuximab-resistant LIM1215 cells both in vitro and in vivo. This study identifies MCT1-dependent lactate utilization as a clinically actionable, metabolic vulnerability to overcome KRAS-mutant-mediated acquired resistance to anti-EGFR therapy in CRC.

Abstract 4728: Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer

Abstract Background: Cetuximab, an epidermal growth factor receptor (EGFR)-targeting IgG1 monoclonal antibody (mAb), results in beneficial, yet limited, clinical improvement in metastatic colorectal cancer (mCRC) patients with wild-type (WT) KRAS gene. Numerous studies have shown that circRNAs are aberrantly expressed in tumors and play crucial roles in tumor growth and metastasis in various human malignancies, including colorectal cancer (CRC). One such circRNA, circ-EGFR, has been identified as having associations with tumor progression and chemotherapy response in diverse cancer types. However, exploring the functional role and underlying mechanisms of circ-EGFR in the context of the response to EGFR-targeted therapy may offer new therapeutic avenues for mCRC. Methods: The expression level of circ-EGFR (hsa_circ_0080229) was evaluated in cetuximab-sensitive and resistant RAS and BRAF WT CRC cell lines by using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The biological function of circ-EGFR was investigated by loss- and gain-of-function assays in cultured cells and an animal model. The potential targets of circ-EGFR were identified through in-silico analysis, and the circ-EGFR/miR-942-3p/GAS1 axis pathway was verified using the luciferase reporter assay and RT-qPCR. The association of circ-EGFR expression with clinical prognosis in mCRC patients with different cetuximab responses was analyzed by Kaplan-Meier survival analysis. Results: The expression of circ-EGFR was remarkedly higher in sensitive (DiFi and SW48) compared with resistant cell lines (CaCO2 and SNU-C1) after exposure of cells to cetuximab (sensitive vs. resistant: p < 0.001). Biologically, genetic depletion of circ-EGFR significantly enhanced cell proliferation, migration [DiFi: sh-circ-EGFR vs. shNC: Foldchange (FC) = 1.97, p < 0.001; SW48: FC = 1.96, p < 0.001], and invasion (DiFi: sh-circ-EGFR vs. shNC: FC = 2.23, p < 0.001; SW48: FC = 1.78, p < 0.001) after 24-48 hours of cetuximab treatment in DiFi and SW48 cell lines, and its overexpression suppressed tumor growth in vivo under cetuximab treatment. Mechanically, we identified circ-EGFR as a sponge for miR-942-3p to regulate the expression of Growth Arrest Specific 1 (GAS1), which promoted sensitivity to cetuximab treatment in CRC. Notably, our findings identified circ-EGFR as a promising therapeutic indicator in clinical applications. Conclusion: Our study illustrated that circ-EGFR plays a vital role in affecting cetuximab efficacy by regulating the miR-942-3p/GAS1 axis in CRC. This holds promise for improving patient selection and management in this malignancy. Citation Format: Silei Sui, Caiming Xu, Yuan Li, Wenyi Wei, Joan Maurel, Man Li, Ajay Goel. Circ-EGFR enhances cetuximab efficacy by regulating miR-942-3p/GAS1 signaling axis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4728.

Triple Targeting of HER Receptors Overcomes Heregulin-mediated Resistance to EGFR Blockade in Colorectal Cancer.

Current treatment options for patients with advanced colorectal cancers include anti-EGFR/HER1 therapy with the blocking antibody cetuximab. Although a subset of patients with KRAS WT disease initially respond to the treatment, resistance develops in almost all cases. Relapse has been associated with the production of the ligand heregulin (HRG) and/or compensatory signaling involving the receptor tyrosine kinases HER2 and HER3. Here, we provide evidence that triple-HER receptor blockade based on a newly developed bispecific EGFR×HER3-targeting antibody (scDb-Fc) together with the HER2-blocking antibody trastuzumab effectively inhibited HRG-induced HER receptor phosphorylation, downstream signaling, proliferation, and stem cell expansion of DiFi and LIM1215 colorectal cancer cells. Comparative analyses revealed that the biological activity of scDb-Fc plus trastuzumab was sometimes even superior to that of the combination of the parental antibodies, with PI3K/Akt pathway inhibition correlating with improved therapeutic response and apoptosis induction as seen by single-cell analysis. Importantly, growth suppression by triple-HER targeting was recapitulated in primary KRAS WT patient-derived organoid cultures exposed to HRG. Collectively, our results provide strong support for a pan-HER receptor blocking approach to combat anti-EGFR therapy resistance of KRAS WT colorectal cancer tumors mediated by the upregulation of HRG and/or HER2/HER3 signaling.

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

Angiotensin-converting Enzyme 2–containing Small Extracellular Vesicles and Exomeres Bind the Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein

U3-1402, a Novel HER3-Targeting Antibody-Drug Conjugate, for the Treatment of Colorectal Cancer.

HER3 is overexpressed in several cancers, including colorectal cancer. Although therapies with anti-HER3 antibodies have been investigated, significant clinical benefits have not been reported. U3-1402 is a novel HER3-antibody-drug conjugate (ADC) composed of the HER3 antibody patritumab and a novel topoisomerase I inhibitor, DX-8951 derivative (DXd). The sensitivity of DXd was evaluated by a growth inhibition assay. The antitumor activity of U3-1402 was evaluated in a murine xenograft model in which its effects on cells, with a range of HER3 expression levels, were compared with those of patritumab alone, irinotecan, control-ADC, and saline. In the growth inhibition assay, all colorectal cancer cell lines were sensitive to DXd. In the tumor xenograft model, significant tumor regression with U3-1402 was observed both in the DiFi cell line (high HER3 expression; KRAS wild type) and in SW620 (high HER3 expression; KRAS mutation), but no treatment effect was observed in Colo320DM (low HER3 expression). Notably, SW620 tumor growth was significantly suppressed with U3-1402 compared with the saline-treated group (P < 0.001) and showed greater activity compared with the irinotecan group. By contrast, patritumab alone, control-ADC, and saline did not significantly differ in tumor growth inhibition. The antitumor activity of U3-1402 was dependent on HER3 expression level, but not on KRAS mutation status. These results support further investigation of development strategies for U3-1402 in patients with HER3-expressing colorectal cancer.

In vivo magnetic resonance imaging of treatment-induced apoptosis

Imaging apoptosis could provide an early and specific means to monitor tumor responses to treatment. To date, despite numerous attempts to develop molecular imaging approaches, there is still no widely-accepted and reliable method for in vivo imaging of apoptosis. We hypothesized that the distinct cellular morphologic changes associated with treatment-induced apoptosis, such as cell shrinkage, cytoplasm condensation, and DNA fragmentation, can be detected by temporal diffusion spectroscopy imaging (TDSI). Cetuximab-induced apoptosis was assessed in vitro and in vivo with cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell lines by TDSI. TDSI findings were complemented by flow cytometry and immunohistochemistry. Cell cycle analysis and flow cytometry detected apoptotic cell shrinkage in cetuximab-treated DiFi cells, and significant apoptosis was confirmed by histology. TDSI-derived parameters quantified key morphological changes including cell size decreases during apoptosis in responsive tumors that occurred earlier than gross tumor volume regression. TDSI provides a unique measurement of apoptosis by identifying cellular characteristics, particularly cell shrinkage. The method will assist in understanding the underlying biology of solid tumors and predict tumor response to therapies. TDSI is free of any exogenous agent or radiation, and hence is very suitable to be incorporated into clinical applications.

Azithromycin enhances anticancer activity of TRAIL by inhibiting autophagy and up-regulating the protein levels of DR4/5 in colon cancer cells in vitro and in vivo

BackgroundAzithromycin is a member of macrolide antibiotics, and has been reported to inhibit the proliferation of cancer cells. However, the underlying mechanisms are not been fully elucidated. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively targets tumor cells without damaging healthy cells. In the present study, we examined whether azithromycin is synergistic with TRAIL, and if so, the underlying mechanisms in colon cancers.MethodsHCT-116, SW480, SW620 and DiFi cells were treated with azithromycin, purified TRAIL, or their combination. A sulforhoddamine B assay was used to examine cell survival. Apoptosis was examined using annexin V-FITC/PI staining, and autophagy was observed by acridine orange staining. Western blot analysis was used to detect protein expression levels. In mechanistic experiments, siRNAs were used to knockdown death receptors (DR4, DR5) and LC-3B. The anticancer effect of azithromycin and TRAIL was also examined in BALB/c nude mice carrying HCT-116 xenografts.ResultsAzithromycin decreased the proliferation of HCT-116 and SW480 cells in a dose-dependent manner. Combination of azithromycin and TRAIL inhibited tumor growth in a manner that could not be explained by additive effects. Azithromycin increased the expressions of DR4, DR5, p62 and LC-3B proteins and potentiated induction of apoptosis by TRAIL. Knockdown of DR4 and DR5 with siRNAs increased cell survival rate and decreased the expression of cleaved-PARP induced by the combination of azithromycin and TRAIL. LC-3B siRNA and CQ potentiated the anti-proliferation activity of TRAIL alone, and increased the expressions of DR4 and DR5.ConclusionThe synergistic antitumor effect of azithromycin and TRAIL mainly relies on the up-regulations of DR4 and DR5, which in turn result from LC-3B-involved autophagy inhibition.

Efficacy and toxicity of HLX07 as a new anti-EGFR monoclonal antibody for epithelial cancers.

e14078 Background: EGFR is a validated target for anti-cancer drug development. Several anti-EGFR monoclonal antibodies (mAb) are used in clinics, but associated with a variety of toxicities. A new anti-EGFR mAb might offer better efficacy with less toxicities. Methods: we have developed a new humanized anti-EGFR mAb, HLX07, through engineering of Fab portion and improvement of glycosylation profile and tested in in vitro assays, in vivo xenograft animal models and primate pharmacokinetic (PK) and toxicokinetic (TK) studies. Results: HLX07 exerted potent antibody-dependent, cell-mediated cytotoxicity in vitro. It specifically binds to monkey and human but not rodent EGFR. HLX07 has better binding affinity to human EGFR, compared with cetuximab (KD 1.43x10-10 vs. 2.62x10-10). HLX07 completely inhibited the phosphorylation of EGFR in H292 cells at ≥0.1 nM, The IC50 of HLX07 for DiFi and H292 cells were 54.2 and 23.9 ng/mL, respectively, compared with 63.7 and 37.2 ng/mL by cetuximab, respectively. In vivo xenogenic studies also demonstrated that HLX07 significantly inhibited the growth of H292, A432 and WiDi cells in a dose-dependent manner. Xenogenic studies also showed that HLX07 used with gemcitabine and/or cisplatin was more active than either agent alone. HLX07 was more effective than cetuximab in inhibiting the growth of FaDu cells when used with local radiotherapy in vivo. Single-dose and 13-week repeat-dose GLP pharmacokinetic and toxicokinetic studies in cynomulgus monkeys up to 60 mg/kg/wk shows minimal-to-mild toxicities (see table). Conclusions: The detailed preclinical characterization indicates that HLX07 has better efficacy and less toxicity than cetuximab. A phase 1 first-in-human study is ongoing (NCT02648490). [Table: see text]

Heat-shock protein 27 (HSP27, HSPB1) is synthetic lethal to cells with oncogenic activation of MET, EGFR and BRAF.

The small heat‐shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene‐addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET‐addicted EBC‐1 lung cancer cells, epidermal growth factor receptor (EGFR)‐addicted colorectal carcinoma (CRC) DiFi cells and BRAF‐addicted CRC COLO205 and OXCO‐1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET‐addicted gastric carcinoma MKN45 and EGFR‐addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro‐apoptotic proteins of the BCL2 family and of active caspase‐3 and lamin B. Together, these data suggest that oncogene‐addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents.

Identification and validation of COX-2 as a co-target for overcoming cetuximab resistance in colorectal cancer cells

Cetuximab, an epidermal growth factor receptor (EGFR)-blocking antibody, was approved for treatment of metastatic colorectal cancer over a decade ago; however, patients' responses to cetuximab vary substantially due to intrinsic and acquired resistance to cetuximab. Here, we report our findings using Affymetrix HG-U133A array to examine changes in global gene expression between DiFi, a human colorectal cancer cell line that is highly sensitive to cetuximab, and two other cell lines: DiFi5, a DiFi subline with acquired resistance to cetuximab, and DiFi-AG, a DiFi subline with acquired resistance to the EGFR tyrosine kinase inhibitor AG1478 but sensitivity to cetuximab. We identified prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2 (COX-2), as the gene with the greatest difference between the cetuximab-resistant DiFi5 cells and the cetuximab-sensitive DiFi cells and DiFi-AG cells. Reverse transcription polymerase chain reaction and Western blotting validated upregulation of COX-2 in DiFi5 but not in DiFi or DiFi-AG cells. We developed COX-2 knockdown stable clones from DiFi5 cells and demonstrated that genetic knockdown of COX-2 partially re-sensitized DiFi5 cells to cetuximab. We further confirmed that cetuximab in combination with a COX-2 inhibitor led to cell death via apoptosis or autophagy not only in DiFi5 cells but also in another colorectal cancer cell line naturally resistant to cetuximab. Our findings support further evaluation of the strategy of combining cetuximab and a COX-2 inhibitor for treatment of metastatic colorectal cancer.

Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting

Exosomes are small, 40–130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes “conformationally active” EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours.

Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression, HER-2/HER-3 signalling and sensitivity to pan HER blockers.

Background:The human epidermal growth factor receptor (EGFR) is an important target for cancer treatment. Currently, only the EGFR antibodies cetuximab and panitumumab are approved for the treatment of patients with colorectal cancer. However, a major clinical challenge is a short-term response owing to development of acquired resistance during the course of the treatment.Methods:In this study, we investigated the molecular mechanisms underlying development of acquired resistance in DiFi colorectal cancer cells to the anti-EGFR mAb ICR62 (termed DiFi62) and to the small molecule tyrosine kinase inhibitor (TKI) gefitinib (termed DiFiG) using a range of techniques.Results:Compared with the findings from parental DiFi and DiFiG cells, development of acquired resistance to anti-EGFR mAb ICR62 in DiFi62 cells was accompanied by an increase in cell surface EGFR and increased phosphorylation of HER-2 and HER-3. Interestingly, DiFi62 cells also acquired resistance to treatment with anti-EGFR mAbs cetuximab and ICR61, which bind to other distinct epitopes on the extracellular domain of EGFR, but these cells remained equally sensitive as the parental cells to treatment with pan-HER inhibitors such as afatinib.Conclusions:Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed.

Antitumor Activity of HM781-36B, alone or in Combination with Chemotherapeutic Agents, in Colorectal Cancer Cells.

PurposeHM781-36B is a novel and irreversible pan-human epidermal growth factor receptor (HER) inhibitor with TEC cytoplasmic kinase inhibition. The aim of this study is to evaluate the antitumor activity and mechanism of action for HM781-36B in CRC cell lines.Materials and MethodsThe CRC cell lines were exposed to HM781-36B and/or oxaliplatin (L-OHP), 5-fluorouracil (5-FU), SN-38. The cell viability was examined by Cell Titer-Glo luminescent cell viability assay kit. Change in the cell cycle and protein expression was determined by flow cytometry and immunoblot analysis, respectively. Synergism between 2 drugs was evaluated by the combination index.ResultsThe addition of HM781-36B induced potent growth inhibition in both DiFi cells with EGFR overexpression and SNU-175 cells (IC50 = 0.003 and 0.005 μM, respectively). Furthermore, HM781-36B induced G1 arrest of the cell cycle and apoptosis, and reduced the levels of HER family and downstream signaling molecules, pERK and pAKT, as well as nonreceptor/cytoplasmic tyrosine kinase, BMX. The combination of HM781-36B with 5-FU, L-OHP, or SN-38 showed an additive or synergistic effect in most CRC cells.ConclusionThese findings suggest the potential roles of HM781-36B as the treatment for EGFR-overexpressing colon cancer, singly or in combination with chemotherapeutic agents. The role of BMX expression as a marker of response to HM781-36B should be further explored.

Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

The anti-HER3 antibody patritumab abrogates cetuximab resistance mediated by heregulin in colorectal cancer cells.

We previously showed that tumor-derived heregulin, a ligand for HER3, is associated with both de novo and acquired resistance to cetuximab. We have now examined whether patritumab, a novel neutralizing monoclonal antibody to HER3, is able to overcome such resistance. Human colorectal cancer (DiFi) cells that are highly sensitive to cetuximab were engineered to stably express heregulin by retroviral infection, and the effects of cetuximab and patritumab on the resulting DiFi-HRG cells were examined. DiFi-HRG cells released substantial amounts of heregulin and showed resistance to cetuximab. Cetuximab alone inhibited EGFR and ERK phosphorylation in DiFi-HRG cells, but it had no effect on the phosphorylation of HER2, HER3, or AKT, suggesting that sustained AKT activation by HER2 and HER3 underlies cetuximab resistance in these cells. In contrast, patritumab in combination with cetuximab markedly inhibited the phosphorylation of EGFR, HER2, HER3, ERK, and AKT. The combination therapy also inhibited the growth of DiFi-HRG tumor xenografts in nude mice to a greater extent than did treatment with either drug alone. Activation of HER2-HER3 signaling associated with the operation of a heregulin autocrine loop confers resistance to cetuximab, and patritumab is able to restore cetuximab sensitivity through inhibition of heregulin-induced HER3 activation.

Abstract 1844: Novel HER3 neutralizing antibody, patritumab abrogates cetuximab resistance mediated by a heregulin-autocrine loop in colorectal cancer

Abstract Purpose Acquired resistance to cetuximab is a major problem in the treatment of colorectal cancers. We previously showed that the HER3/HER4 ligand heregulin induced resistance to cetuximab by increasing the association between HER2 and HER3, and subsequent activation of downstream targets. We have now investigated strategies to overcome such resistance, using patritumab, a novel neutralizing HER3 antibody. Experimental design We established DiFi cells that stably express heregulin (DiFi-HRG cells) as a result of retroviral infection, and examined the antitumor effect of cetuximab, patritumab, and the combination of both in these cells in vitro and vivo. Results DiFi-HRG cells secreted large amount of heregulin and exhibited resistance to cetuximab. Cetuximab alone down-regulated ERK phosphorylation, whereas it had no effect on AKT phosphorylation, suggesting that continuous AKT activation underlain underlies resistance to cetuximab in these cells. In contrast, Patritumab in combination with cetuximab markedly reduced the level of phosphorylation of HER2 HER3 and AKT, as well as ERK, and induced apoptosis that was accompanied by upregulation of BIM and by downregulation of survivin in vitro. Furthermore, the combination therapy also markedly inhibited the growth of DiFi-HRG tumors in nude mice to a greater extent than did treatment with either drug alone. Conclusion Our data indicate that HER2/HER3 signaling activated by a heregulin-autocrine loop confers resistance to cetuximab, and that patritumab restored sensitivity to cetuximab by inhibition of HER3 phosphorylation that is stimulated by heregulin. Citation Format: Hisato Kawakami, Isamu Okamoto, Kimio Yonesaka, Kunio Okamoto, Kiyoko Kuwata, Yume Morita, Haruka Yamaguchi, Kazuto Nishio, Kazuhiko Nakagawa. Novel HER3 neutralizing antibody, patritumab abrogates cetuximab resistance mediated by a heregulin-autocrine loop in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1844. doi:10.1158/1538-7445.AM2014-1844

Neu3 Activity Enhances Egfr Activation Without Affecting Egfr Expression

ABSTRACT Aim: The plasma membrane associated sialidase NEU3 is often deregulated in colorectal cancer (CRC) and was demonstrated to coimmunoprecipitate in HeLa cells with the epidermal growth factor receptor (EGFR), the molecular target of the recent therapies based on monoclonal antibodies. Elucidating this correlation may provide novel understandings in cancer biology and may represent a new basis to develop innovative therapeutic approaches. Therefore, our aim was to evaluate the effect of NEU3 deregulation on EGFR expression and activation in CRC cell lines. Methods: NEU3 and EGFR expression was evaluated by Real-time PCR (Q-PCR) in 13 human CRC cell lines, as well as in DiFi cells. Regulation of EGFR pathway by NEU3 activity was studied using NEU3 wild-type (wt) and double-mutant (encoding for NEU3 inactivated protein) vectors transiently transfected in DiFi and SW480 cell lines. EGFR gene and mutational status were assessed by FISH and direct sequencing. Western Blot analysis was performed to study expression and phosphorylation status of EGFR downstream proteins. EGFR sialylation levels were evaluated by lecting binding assay using biotinilated SNA (Sambucus nigra agglutinin) and avidin-horseradish peroxidase. Cell viability was assessed by MTT assay. Results: We observed up-regulation of NEU3 mRNA levels and EGFR activation in all the investigated cell lines, independent of EGFR mRNA/protein expression or gene status. Transfection assays confirmed that only NEU3 wt enhanced EGFR activation without affecting its expression. Moreover, the level of EGFR sialylation was reduced in cells overexpressing the NEU3 wt sialidase, strongly suggesting that EGFR sialylation is regulated by NEU3. MTT test's results showed a significant increase of cell viability (p Conclusions: Since NEU3 reveals to be overexpressed in the totality of cancer cell lines analyzed and its activity regulates the EGFR pathway, we assumed this sialidase as a good and innovative marker for diagnosis and therapy of CRC. Disclosure: All authors have declared no conflicts of interest.

Abstract A62: p38MAPK implication in cetuximab response.

Abstract Cetuximab is used in colorectal cancer (CRC), as targeted therapy against the Epithelial Growth Factor receptor (EGFR), in association with chemotherapy (5-FU and irinotecan). Its binding inhibits signalling pathways downstream to the receptor leading to a decrease in proliferation and surviving. In KRAS mutated CRC patients, cetuximab is ineffective, and half of KRAS Wild Type (WT) patients does not respond to cetuximab either. Thus, a better knowledge of cetuximab mechanism of action will help to improve response rate. We have previously shown that activation of the MAP Kinase p38 (p38MAPK) induces irinotecan resistance in vitro and in vivo. The aim of our project is to determine, in KRAS WT colorectal cells, if p38MAPK is involved as well in the cetuximab effect. Experiments were done on two KRAS WT CRC cell lines which respond differently to cetuximab: Caco2 cells (30% of survival inhibition, p53 mutated) and DiFi cells (80% of survival inhibition, p53 Wild Type). Cytotoxic experiments combining cetuximab treatment and inhibition of p38MAPK by a pharmacological inhibitor (SB202190) were performed. We assessed apoptosis and inhibition of proliferation by FACS analysis of cell cycle and DNA synthesis. In addition, Erk, Bim and p27 were analysed by Western Blot. Our results showed that inhibition of p38MAPK by SB202190 enhances cetuximab cytotoxic effect in Caco2 cells but impairs it in DiFi cells. We also observed that inhibition of p38MAPK decreases cetuximab induced apoptosis (from 40% of cells in subG1 to 15%) and anti-proliferative effect (from 10% EdU positive cells to 40%) in DiFi cells. The prevention of cell death by SB202190 in cetuximab treated DiFi cells could be explained by Erk pathway activation and the decrease of p27 and Bim respectively involved in cellular proliferation and mitochondrial apoptosis. We have shown that genetic inhibition of p53 also prevents cetuximab cytotoxicity in DiFi cells and we observed a synergistic effect with p38MAPK inhibition leading to higher impairment of cetuximab effect. Our results have shown the same inhibiting effect of SB202190 on the cytotoxic effect of two tyrosine-kinase inhibitors targeting EGFR (lapatinib and erlotinib) in DiFi cells indicating that p38MAPK implication is linked to inhibition of EGFR kinase activity not to cetuximab only. We have shown that p38MAPK is involved in response to the inhibition of EGFR activity. However, it seems to have different role according to the cell line used and genetic background (p53 status). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A62. Citation Format: Laetitia Marzi, Eve Combès, Nadia Vezzio-Vié, Gaelle Thomas, Christel Larbouret, Laetitia Linares, Clara Montagut, Mar Iglesias, Vincent Denis, Maguy Del Rio, Pierre Martineau, Céline Gongora. p38MAPK implication in cetuximab response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A62.

Abstract 5631: Acquired resistance to anti-EGFR mAb ICR62 or afatinib is accompanied by upregulation of the EGFR in colorectal cancer .

Abstract The aberrant expression and activation of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (also called HER or erbB family) have been implicated in a wide range of cancers. These receptors have emerged as important therapeutic targets in human cancers. To date, of the two classes of HER inhibitors (i.e. monoclonal antibodies (mAbs) and small molecule tyrosine kinase inhibitors (TKIs), cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer. However, many patients develop resistance to the EGFR inhibitors and consequently the duration of response to these treatments is often short. In this study on colorectal cancer cells, we evaluated whether there was any association between the expression levels of the HER family members, putative colorectal cancer stem cell (CSC) markers CD133 and CD44, and ABC transporter and the development of resistance to anti-EGFR mAb ICR62 or afatinib, an irreversible erbB blocker. A wide range of techniques were employed in this study including FACS analysis, Western blot analysis and SRB assay on the EGFR over-expressing human colorectal tumour cell line DiFi, which is highly sensitive to treatment with mAb ICR62 (IC50=1.2 nM) and afatinib (IC50=10 nM). Continuous exposure of DiFi cells to increasing doses of mAb ICR62 or afatinib for 6-10 months resulted in the emergence of ICR62 variant DiFi cells (DiFi62) and afatinib-variant DiFi cells (DiFiAf) with IC50 values of 209 nM and 35 nM respectively. Interestingly, we found that ICR62 resistant DiFi cells (DiFi62) also become less sensitive to treatment with 5FU, oxaloplatin and irinotecan, but remained highly sensitive to treatment with afatinib (IC50=8 nM). In contrast to DiFi62 cells, afatinib-resistant DiFiAf cells were less sensitive to treatment with 5FU and irinotecan, but like parental DiFi cells remained highly sensitive to treatment with ICR62 and oxaliplatin. Of the HER family members, there was only upregulation of EGFR in both DiFi62 and DiFiAf cells, but no changes in the expression levels of other members of the HER family or CD133. In contrast, the expression of CD44, the other putative colorectal CSC marker, was found to be downregulated by around 38% in DiFiAf cells. Of the ABC transporters, there was an increase in the expression of p-glycoprotein in both DiFi62 and DiFiAf cells and an increased expression of both MRP2 and ABCG2 in DiFi62, but not in DiFiAf cells. Taken together, our results suggest that colorectal tumour cells which acquire resistance to anti-EGFR mAb ICR62 remain sensitive to treatment with the irreversible erbB blocker afatinib and vice versa, suggesting that mechanisms underlying the antitumor activity of ICR62 and afatinib are different in these cells. In addition, upregulation of the EGFR upon treatment with these inhibitors may contribute to resistance. Citation Format: Said Abdullah Khelwatty, Sharadah Essapen, Alan M. Seddon, Zhen Fan, Helmout Modjtahedi. Acquired resistance to anti-EGFR mAb ICR62 or afatinib is accompanied by upregulation of the EGFR in colorectal cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5631. doi:10.1158/1538-7445.AM2013-5631