LIM1215 Cells Research Articles

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.

BRAF V600E-induced distinct DNA damage response defines the therapeutic potential of p53 activation for TP53 wild-type colorectal cancer.

BRAF V600E, one of the most frequent mutations in the MAPK pathway, confers poor prognosis to colorectal cancers (CRCs), partly because of chemotherapeutic resistance. Oncogene-induced DNA damage responses (DDRs) that primarily activate p53 are important mechanistic barriers to the malignant transformation of cells; however, the mechanism underlying this impairment in cancer remains unknown. Here, we evaluated the responses of BRAFV600E-induced DDRs in two CRC cell lines, SW48 and LIM1215, both of which harbor wild-type TP53, KRAS, and BRAF. BRAFV600E transduction exhibited distinct phenotypes in these cells: SW48 cell proliferation markedly decreased, whereas that of LIM1215 increased. BRAFV600E expression induced the activation of oncogene-induced DDR signaling in SW48 cells, but not in LIM1215 cells, whereas chemotherapeutic agents similarly activated DDRs in both cell lines. Knockdown experiments revealed that these responses in SW48 cells were mediated by p53-p21 pathway activation. Comet assay (both alkaline and neutral) revealed that BRAFV600E increased single-strand breaks to the same extent in both cell lines; however, in case of LIM1215 cells, it only facilitated double-strand breaks. Furthermore, the proliferation of LIM1215 cells, wherein no oncogene-induced DDRs occurred, was synergistically inhibited upon MDM2 inhibitor-mediated p53 activation combined with MEK inhibition. Taken together, these distinct DDR signaling responses highlight the novel characteristics of BRAFV600E-mutated CRC cells and define the therapeutic potential of p53 activation combined with MAPK inhibition against TP53 wild-type CRC harboring a BRAFV600E mutation.

Discovery of cancer-preventive juices reactivating RB functions.

Recent advances have been achieved in the genetic diagnosis and therapies against malignancies due to a better understanding of the molecular mechanisms underlying carcinogenesis. Since active preventive methods are currently insufficient, the further development of appropriate preventive strategies is desired. We searched for drinks that reactivate the functions of tumor-suppressor retinoblastoma gene (RB) products and exert anti-inflammatory and antioxidant effects. We also examined whether lactic acid bacteria increased the production of the cancer-specific anti-tumor cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in human, and examined whether the RB-reactivating drinks with lactic acid bacteria decreased azoxymethane-induced rat colon aberrant crypt foci (ACF) and aberrant crypts (ACs) in vivo. Kakadu plum juice and pomegranate juice reactivated RB functions, which inhibited the growth of human colon cancer LIM1215 cells by G1 phase arrest. These juices also exerted anti-inflammatory and antioxidant effects. Lactiplantibacillus (L.) pentosus S-PT84 was administered to human volunteers and increased the production of TRAIL. In an in vivo study, Kakadu plum juice with or without pomegranate juice and S-PT84 significantly decreased azoxymethane-induced rat colon ACF and ACs. RB is one of the most important molecules suppressing carcinogenesis, and to the best of our knowledge, this is the first study to demonstrate that natural drinks reactivated the functions of RB. As expected, Kakadu plum juice and pomegranate juice suppressed the growth of LIM1215 cells by reactivating the functions of RB, and Kakadu plum juice with or without pomegranate juice and S-PT84 inhibited rat colon ACF and ACs. Therefore, this mixed juice has potential as a novel candidate for cancer prevention.

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

A33 antibody-functionalized exosomes for targeted delivery of doxorubicin against colorectal cancer

Exosomes have emerged as a promising drug carrier with low immunogenicity, high biocompatibility and delivery efficiency. Here in, we isolated exosomes from A33-positive LIM1215 cells (A33-Exo) and loaded them with doxorubicin (Dox). Furthermore, we coated surface-carboxyl superparamagnetic iron oxide nanoparticles (US) with A33 antibodies (A33Ab-US), expecting that these A33 antibodies on the surface of the nanoparticles could bind to A33-positive exosomes and form a complex (A33Ab-US-Exo/Dox) to target A33-positive colon cancer cells. The results showed that A33Ab-US-Exo/Dox had good binding affinity and antiproliferative effect in LIM1215 cells, as shown by increased uptake of the complex. In vivo study showed that A33Ab-US-Exo/Dox had an excellent tumor targeting ability, and was able to inhibit tumor growth and prolong the survival of the mice with reduced cardiotoxicity. In summary, exosomes functionalized by targeting ligands through coating with high-density antibodies may prove to be a novel delivery system for targeted drugs against human cancers.

LRF inhibits p53 expression in colon cancer cells via modulating DAP5 activity.

The p53 protein plays a critical role in suppression of tumour growth; its regulation is not fully understood. Leukaemia/lymphoma-related factor (LRF) promotes tumour cell growth. This study tests a hypothesis that LRF inhibits p53 expression in colon cancer cells. In this study, human colon cancer cell lines, LIM1215 and HCT116 cells, were used. The expression of LRF and p53 in the cells was analysed by quantitative reverse transcription polymerase chain reaction and Western blotting. We observed that the expression of protease-activated receptor 2 (PAR2) was detected in both LIM1215 and HCT116 human colon cancer cells. Activation of PAR2 increased the expression of LRF and inhibited the p53 expression in the cancer cells. We also detected a complex of LRF and DAP5, one of the p53 gene transcription factors. The interaction of LRF and DAP5 resulted in the repression of p53 expression in the colon cancer cells. In conclusion, PAR2 activation increases the expression of LRF in colon cancer cells, which interacts with DAP5 to repress the p53 expression. Leukaemia/lymphoma-related factor may be a novel target in the treatment of colon cancer.

Abstract 26: Panitumumab interacts with TAS-102 leading to combinational anti-cancer effects by blocking EGFR-mediated tumor response to trifluridine

Abstract Panitumumab is a monoclonal antibody raised against the human epidermal growth factor receptor (EGFR). TAS-102 is a novel chemotherapeutic agent containing trifluridine (FTD) as the active cytotoxic component. Both panitumumab and TAS-102 have been approved for the treatment of metastatic colorectal cancer (mCRC). In this study, we show the mechanism underlying the anti-cancer effects of the panitumumab/TAS-102 combination in preclinical models. Co-treatment with panitumumab and FTD exerted additive and synergistic anti-proliferative effects in LIM1215 and SW48 colon cancer cells, respectively. Consistent with the in vitro effects, panitumumab/TAS-102 combination led to tumor regression in LIM1215 and COL-01-JCK colon cancer patient-derived xenograft models. In LIM1215 cells, FTD induced ERK/Akt/STAT3 phosphorylation and subsequent serine/threonine phosphorylation of EGFR, while it had no effects on EGFR tyrosine phosphorylation. Panitumumab and the tyrosine kinase inhibitor erlotinib reduced the basal level of EGFR tyrosine phosphorylation and reversed the FTD-induced ERK/Akt/STAT3 and EGFR serine/threonine phosphorylation. These results suggested that FTD together with the basal activity of the EGFR tyrosine kinase induced downstream pro-survival signaling through ERK/Akt/STAT3. Collectively, we propose that panitumumab interacts with FTD by targeting EGFR-mediated adaptive responses, thereby exerting anti-cancer effects in combination with TAS-102. These preclinical findings provide a compelling rationale to evaluate anti-EGFR antibodies combined with TAS-102 against mCRC. Citation Format: Kazuhide Nakamura, Yuji Baba, Toshiya Tamura, Yoshihiko Satoh, Masamitsu Gotou, Hiroshi Sawada, Shunsuke Ebara, Kazunori Shibuya, Jumpei Soeda. Panitumumab interacts with TAS-102 leading to combinational anti-cancer effects by blocking EGFR-mediated tumor response to trifluridine [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 26. doi:10.1158/1538-7445.AM2017-26

Panitumumab interaction with TAS-102 leads to combinational anticancer effects via blocking of EGFR-mediated tumor response to trifluridine.

Panitumumab is a monoclonal antibody developed against the human epidermal growth factor receptor (EGFR). TAS‐102 is a novel chemotherapeutic agent containing trifluridine (FTD) as the active cytotoxic component. Both panitumumab and TAS‐102 have been approved for the treatment of metastatic colorectal cancer. In this study, we revealed the mechanism underlying the anticancer effects of panitumumab/TAS‐102 combination using preclinical models. Panitumumab/FTD cotreatment showed additive antiproliferative effects in LIM1215 and synergistic antiproliferative effects in SW48 colon cancer cells. Consistent with the in vitro effects, panitumumab/TAS‐102 combination caused tumor regression in LIM1215 and COL‐01‐JCK colon cancer patient‐derived xenograft models. In LIM1215 cells, FTD induced extracellular signal‐regulated kinase (ERK)/protein kinase B (AKT)/signal transducer and activator of transcription 3 (STAT3) phosphorylation and subsequent serine/threonine phosphorylation of EGFR, while it had no effects on EGFR tyrosine phosphorylation. Panitumumab and the tyrosine kinase inhibitor erlotinib reduced the basal level of EGFR tyrosine phosphorylation and reversed FTD‐induced ERK/AKT/STAT3 and EGFR serine/threonine phosphorylation. These results suggested that FTD in combination with the basal activity of EGFR tyrosine kinase induced downstream prosurvival signaling through ERK/AKT/STAT3 phosphorylation. Collectively, we propose that panitumumab interacts with FTD by targeting EGFR‐mediated adaptive responses, thereby exerting anticancer effects when used in combination with TAS‐102. These preclinical findings provide a compelling rationale for evaluating the combination of anti‐EGFR antibodies with TAS‐102 against metastatic colorectal cancer.

Methods of selecting combination therapy for colorectal cancer patients: a patent evaluation of US20160025730A1

ABSTRACTIntroduction: Colorectal cancer (CRC) is the fourth most common cancer worldwide. Targeted therapy drugs (TTDs) are a valid treatment, epithelial growth factor receptor (EGFR) inhibitors being one of the most commonly used for CRC patients. However, this treatment is only useful for patients with wild-type KRAS (wtKRAS) and is effective only on about 40 to 60% of this subset due to the high plasticity of ErbB network.Areas covered: The invention proposes the use of ErbB protein levels and ErbB receptor dimer formation as biomarkers for selecting, predicting and monitoring CRC patients showing sensitivity to the action of EGFR inhibitors to benefit from the combination therapy of EGFR and HER2 inhibitors. The in vitro data on Lim1215 cells suggest the over-activation of HER3 signaling pathway in response to the use of EGFR inhibitors on monotherapy; the use of HER2 or HER3 or MEK inhibitors in combination with EGFR inhibitors reversed this activation.Expert opinion: To assess the clinical applicability of this invention, further studies are needed since the conclusions are derived solely based on the data obtained from only one CRC cell line (Lim1215). Furthermore, other biofactors/mutations should be considered to assure the potential benefits of the combination therapies proposed.

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.

Comparative N-Glycan Profiling of Colorectal Cancer Cell Lines Reveals Unique Bisecting GlcNAc and α-2,3-Linked Sialic Acid Determinants Are Associated with Membrane Proteins of the More Metastatic/Aggressive Cell Lines

Advances in colorectal cancer (CRC) diagnosis will be enhanced by development of more sensitive and reliable methods for early detection of the disease when treatment is more effective. Because many known disease biomarkers are membrane-bound glycoproteins with important biological functions, we chose to compare N-glycan profiles of membrane proteins from three phenotypically different CRC cell lines, LIM1215, LIM1899, and LIM2405, representing moderately differentiated metastatic, moderately differentiated primary, and poorly differentiated (aggressive) primary CRC cell lines, respectively. The N-glycan structures and their relative abundances were determined as their underivatized reduced forms, using porous graphitized carbon LC-ESI-MS/MS. A key observation was the similar N-glycan landscape in these cells with the dominance of high mannose type glycan structures (70-90%) in all three cell lines, suggesting an incomplete glycan processing. Importantly, unique glycan determinants such as bisecting N-acetylglucosamine were observed at a high level in the metastatic LIM1215 cells, with some expressed in the moderately differentiated LIM1899, while none were detected in the poorly differentiated LIM2405 cells. Conversely, α-2,3-sialylation was completely absent in LIM1215 and LIM1899 and present only in LIM2405. RNA-Seq and lectin immunofluorescence data correlated well with these data, showing the highest upregulation of Mgat3 and binding with PHA-E in LIM1215. Downregulation of Man1α1 and Mgat1 in LIM1215 also coincided with the higher degree of incomplete N-glycan processing and accumulation of high mannose type structures as well as bisecting N-glycans when compared to the other two cell lines. This study provides a comprehensive analysis of the membrane N-glycome in three CRC cell lines and identifies N-glycosylation differences that correlate with the histological and pathological features of the cell lines. The unique glycosylation phenotypes may therefore serve as a molecular feature to differentiate CRC disease stages.

Sulindac modulates secreted protein expression from LIM1215 colon carcinoma cells prior to apoptosis

Colorectal cancer (CRC) is a major cause of mortality in Western populations. Growing evidence from human and rodent studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) cause regression of existing colon tumors and act as effective chemopreventive agents in sporadic colon tumor formation. Although much is known about the action of the NSAID sulindac, especially its role in inducing apoptosis, mechanisms underlying these effects is poorly understood. In previous secretome-based proteomic studies using 2D-DIGE/MS and cytokine arrays we identified over 150 proteins released from the CRC cell line LIM1215 whose expression levels were dysregulated by treatment with 1mM sulindac over 16h; many of these proteins are implicated in molecular and cellular functions such as cell proliferation, differentiation, adhesion, angiogenesis and apoptosis (Ji et al., Proteomics Clin. Appl. 2009, 3, 433–451). We have extended these studies and describe here an improved protein/peptide separation strategy that facilitated the identification of 987 proteins and peptides released from LIM1215 cells following 1mM sulindac treatment for 8h preceding the onset of apoptosis. This peptidome separation strategy involved fractional centrifugal ultrafiltration of concentrated cell culture media (CM) using nominal molecular weight membrane filters (NMWL 30K, 3K and 1K). Proteins isolated in the >30K and 3–30K fractions were electrophoretically separated by SDS-PAGE and endogenous peptides in the 1–3K membrane filter were fractioned by RP-HPLC; isolated proteins and peptides were identified by nanoLC-MS–MS. Collectively, our data show that LIM1215 cells treated with 1mM sulindac for 8h secrete decreased levels of proteins associated with extracellular matrix remodeling (e.g., collagens, perlecan, syndecans, filamins, dyneins, metalloproteinases and endopeptidases), cell adhesion (e.g., cadherins, integrins, laminins) and mucosal maintenance (e.g., glycoprotein 340 and mucins 5AC, 6, and 13). A salient finding of this study was the increased proteolysis of cell surface proteins following treatment with sulindac for 8h (40% higher than from untreated LIM1215 cells); several of these endogenous peptides contained C-terminal amino acids from transmembrane domains indicative of regulated intramembrane proteolysis (RIP). Taken together these results indicate that during the early-stage onset of sulindac-induced apoptosis (evidenced by increased annexin V binding, dephosphorylation of focal adhesion kinase (FAK), and cleavage of caspase-3), 1mM sulindac treatment of LIM1215 cells results in decreased expression of secreted proteins implicated in ECM remodeling, mucosal maintenance and cell–cell-adhesion. This article is part of a Special Issue entitled: An Updated Secretome.

Abstract 1219: Identification of synthetic lethal interactions with the BRAF oncogene in colorectal cancer.

Abstract Background Approximately 8-15% of colorectal (CRC) patients carry an activating mutation in BRAF. This CRC subtype is associated with poor outcome and with resistance, both to chemotherapeutic treatments and to tailored drugs. We recently showed that BRAF (V600E) colon cancers (CCs) have a characteristic gene expression signature (1, 2) which is found also in subsets of KRAS mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this gene signature, referred as “BRAF-like," have a similar poor prognosis irrespective of the presence of the BRAF (V600E) mutation. By using a shRNA-based genetic screen in BRAF mutant CC cell lines we aimed to identify genes and pathways necessary for survival and growth of BRAF mutant CC. Such studies may reveal additional targets for therapy and potentially provide new biomarkers for patient stratification. Method We identified 363 genes that are selectively overexpressed in BRAF mutant tumors as compared to WT2 type tumors, based on gene expression profiles of the PETACC3 (1) and Agendia (1) datasets. The TRC human genome-wide shRNA collection (TRC-Hs1.0) was used to generate a 1815 hairpins sub-library targeting those identified genes (BRAF library). BRAF(V600E) CC cell lines were infected with the BRAF library and screened for shRNAs that cause lethality. LIM1215 CC cell line (WT2) was used as a control. Cells stably expressing the shRNA library were cultured for 13 days, after which shRNAs were recovered by PCR. Deep sequencing was applied to determine the specific depletion of shRNA in BRAF(V600E) cells as compared to LIM1215 cells. Results Candidate genes were identified by using following filtering criteria: depletion in BRAF(V600E) cells by at least 50% and depletion in BRAF(V600E) cells 1,5-fold higher than in control cells with the corresponding p-value to be ≤ 0.1. A total of 34 genes met our criteria of which 6 genes were presented with more than one hairpin and were concordant across the cell lines selected for validation. Conclusion We identified candidate synthetic lethal genes in BRAF mutant CC cell lines. Functional analysis is ongoing. Data will be presented. Citation Format: Loredana Vecchione, Valentina Gambino, Giovanni D'Ario, Tian Sun, Iris Simon, Vlad Popovici, Mauro Delorenzi, Rene’ Bernards, Sabine Tejpar. Identification of synthetic lethal interactions with the BRAF oncogene 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 1219. doi:10.1158/1538-7445.AM2013-1219

Identification of synthetic lethal interactions with the BRAF oncogene in colorectal cancer.

403 Background: Approximately 8-15% of colorectal (CRC) patients carry an activating mutation in BRAF. This CRC subtype is associated with poor outcome and with resistance, both to chemotherapeutic treatments and to tailored drugs. We recently showed that BRAF (V600E) colon cancers (CCs) have a characteristic gene expression signature (1, 2) which is found also in subsets of KRAS mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this gene signature, referred as “BRAF-like”, have a similar poor prognosis irrespective of the presence of the BRAF (V600E) mutation. By using a shRNA-based genetic screen in BRAF mutant CC cell lines we aimed to identify genes and pathways necessary for survival and growth of BRAFmutant CC. Such studies may reveal additional targets for therapy and potentially provide new biomarkers for patient stratification Methods: We identified 363 genes that are selectively overexpressed in BRAF mutant tumors as compared to WT2 type tumors, based on gene expression profiles of the PETACC3 (1) and Agendia (2) datasets. The TRC human genome-wide shRNA collection (TRC-Hs1.0) was used to generate a 1815 hairpins sub-library targeting those identified genes (BRAF library). BRAF(V600E) CC cell lines were infected with the BRAF library and screened for shRNAs that cause lethality. LIM1215 CC cell line (WT2) was used as a control. Cells stably expressing the shRNA library were cultured for 13 days, after which shRNAs were recovered by PCR. Deep sequencing was applied to determine the specific depletion of shRNA in BRAF(V600E) cells as compared to LIM1215 cells Results: Candidate genes were identified by using following filtering criteria: depletion in BRAF(V600E) cells by at least 50% and depletion in BRAF(V600E) cells 1, 5-fold higher than in control cells with the corresponding p-value to be ≤ 0.1. A total of 34 genes met our criteria of which 6 genes were presented with more than one hairpin and were concordant across the cell lines selected for validation. Conclusions: We identified candidate synthetic lethal genes in BRAF mutant CC cell lines. Functional analysis is ongoing. Data will be presented.

P2.09 Preclinical Antitumour Activity of S49076, A Novel Met / Axl / Fgfr Kinase Inhibitor, and Molecular Stratification for Tumour Sensitivity

ABSTRACT Aberrant activity of receptor tyrosine kinases (RTKs) has been associated with tumour progression in a wide variety of human malignancies, making them major drug targets for cancer therapy today. Although, in some instances, specific inhibition of just one of these RTKs suffices for inhibition of tumour progression, in the majority of cases, targeting more than one RTK could be required for therapeutic efficacy. Moreover, increased expression or activation of RTKs is often associated with resistance to standard chemotherapy agents or signal transduction modulators. S49076 is a novel, potent, orally-available, ATP-competitive tyrosine kinase inhibitor of the RTKs MET, AXL and FGFR. S49076 blocks autophosphorylation of these RTKs and their downstream signalling in cells with IC50 values of less than 50 nM in the case of MET and AXL, and at below 200 nM for FGFR1/2/3. In vitro, S49076 inhibits the proliferation of MET- and FGFR2- dependent gastric cancer cells, blocks MET-driven migration of lung carcinoma cells and inhibits colony formation of hepatocarcinoma cells overexpressing FGFR2 and AXL. In vivo, oral administration of S49076 inhibits MET autophosphorylation and tumour growth in subcutaneous GTL-16 human gastric carcinoma and U87-MG human glioblastoma at 6 mg/kg/day. In FGFR2-dependent SNU 16 gastric tumours S49076 inhibits FGFR2 autophosphorylation, downstream signalling and tumour growth at 25 mg/kg/day. In a panel of 53 patient-derived tumours and 14 cell lines of diverse origin growing in three-dimensional in vitro culture, twenty-five (37 %) were found to be sensitive to S49076 at 1 µM or less. In the majority of cases, analysis of the expression, activation and mutation status of MET, AXL, FGFR1/2/3 and other target kinases of S49076 as well as that of major signalling proteins enabled hypotheses to be made concerning the sensitivity or resistance to S49076. Moreover, in many of these resistant tumours, a rationale for association of S49076 with other targeted therapies emerged. Studies to investigate the efficacy of such combined therapies are in progress. Based on these preclinical studies showing a favourable and novel pharmacological profile of S49076, a phase I study is currently underway in patients with advanced solid tumours. Download : Download full-size image Fig. 1 . KRAS mutations mediate acquired resistance to cetuximab in Lim1215 cells. Table 1 . Mutational analysis of the KRAS gene in patients. Patient iD Anti-EGFR resistant tumors KRAS Mutational Status Mutation Percentage Reads3/Events b Patient #1 wt a 0% 0/12123 Patient #2 G13D a 10% 859/8556 Patient #4 G13D a 5.9% 461/7764 Patient #5 G13D a 14.3% 1037/7247 Patient #6 G13D a 8.6% 651/7577 Patient #7 wt a 0% 0/17142 Patient #8 Q61 H b 17.3% 5960/190200 Patient #9 G12D b 0.04% 17/40200 G13D b 0.44% 117/26400 Patient #10 wt b 0%: 0/50300 Patient #11 wt (amplified) b 0% 0/30400 a: 454 b: Beaming

Abstract 1078: ErbB/Pi3K signaling contributes to EGFR ligand expression in colorectal cancer cells

Abstract Background: Cellular expression of EGFR ligands is tightly regulated and is thought to be maintained by an autocrine signaling component in colorectal cancer cells. More importantly, the expression levels of individual ligands have gained recent interest since their expression of their mRNA was found to be linked to anti-EGFR therapy response. Since the expression of these ligands can be regulated by EGFR signaling elicited by EGF, we decided to investigate the contribution of MAPK as well as the PI3K axis, to EREG expression, in K-Ras wild-type colon cancer cells. Methods Colon cancer cells were used to interrogate EGFR downstream signaling. Several inhibitors targeting the ErbBs and the MAPK and PI3K/Akt pathways were used to dissect the pathways downstream of EGFR maintaining ligand mRNA levels in the lim1215 colon cancer cell line. Additionally, we used ER and AR reporter assays to selectively evaluate involvement of the EGFR pathway in transcriptional regulation of the pathway. Consequently, blockade and activation of EGFR pathway members was assessed by Western Blot. Results We show that Ereg and Areg mRNA levels are specifically maintained by erbb1 signaling. Treatment of lim1215 cells with inhibitors blocking Erbb1 (cetuximab) or ErbB1/Erbb2 (lapatinib) did results in near complete inhibition of Ereg and Areg expression in Kras/pi3K wild-type cells, while blockade of ErbB2 alone (which is activated in these cells) with Trastuzumab did not decrease Ereg nor Areg mRNA levels. Downstream of EGFR, we show that MEK signaling is not required for maintenance of EREG and AREG levels in lim1215 cells. Conversely, blockade of PI3K signaling by Akt inhibitors or PI3K inhibitors abolished EREG mRNA levels in these cells. This was reflected by the ability of overexpressed Akt to induce EREG and AREG reporter activity in 293T cells. In contrast, this activation could be rescued by the addition of Akt and PI3K inhibitors, but not MEK inhibitors in cellular media. Conclusions In this work we show that PI3K signaling is required for the transcription of EGFR ligands in colon cancer cells. In this regard, this provides additional insights to the recent findings that link PI3K/Akt signaling to Receptor Tyrosine Kinase expression and activation in cancer cells. Therefore, a global understanding of the signaling networks that control ligand availability and receptor abundance will be crucial in combining ErbB pathway inhibitors in the treatment of kras wild-type colon cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1078. doi:1538-7445.AM2012-1078

Abstract A70: Isogenic K-Ras mutant cancer cells: A novel platform for drug profiling.

Abstract Ras mutations are present in approximately 49% of human colorectal cancers, with mutations in K-Ras G12 and G13 being the most common. Given their high prevalence, understanding how Ras mutations impact response to anticancer agents is crucial to the successful development of novel agents. Recent evidence suggests that not all variants of K-Ras respond in the same way to targeted agents, where some patients with G13D mutations respond better to Cetuximab compared to patients expressing G12V (De Roock, JAMA 2011). Therefore there is a need for better in vitro models to facilitate the characterisation of inhibitor selectivity and efficacy in K-Ras mutant cell systems. We created suites of isogenic X-MAN SW48 and LIM1215 colorectal cancer cells, each of which harbour one of 7 different K-Ras G12 or G13 mutations at their endogenous loci introduced via AAV-mediated homologous recombination. This system allows use of endogenous promoters and has enabled us to create a panel of human cell lines in which the only difference between the cells is the point mutation of interest. This more accurately recapitulates the genomic architecture in human tumors and provides an excellent platform for addressing the impact of K-Ras mutations on responses to therapeutic agents. We selected over 30 targeted agents and cytotoxic drugs based on their clinical relevance to colorectal cancer. We profiled the anti-proliferative activity of each of these across the suite of K-Ras variants in SW48 cells. Strikingly, we found a consistent pattern of resistance towards 5 chemically distinct MEK1/2 inhibitors across the 7 K-Ras variants compared to wild-type cells. These results were confirmed in a second suite of K-Ras mutant lines generated in LIM1215 cells, where clear resistance to MEK inhibitors was observed. To investigate the mechanism of this resistance to MEK inhibitors, we combined the anti-EGFR monoclonal antibody Cetuximab with the MEK inhibitor AZD6244, both compounds being ineffective as single agents in the K-Ras G12D and G12C mutant SW48 cells. Concentrations of Cetuximab as low as 0.01μg/ml re-sensitised previously resistant K-Ras mutant cells to the effects of AZD6244. Given that this dose of Cetuximab was ineffective alone, this result suggests that upregulation of the EGFR axis may be a potential mechanism in compensating for MEK inhibition, and these studies are on-going. In conclusion, we have created two suites of isogenic colorectal cancer cell lines each expressing a different K-Ras G12 or G13 mutation and demonstrated that K-Ras mutations impart resistance to clinical MEK inhibitors. Despite screening over 30 chemotherapeutics covering an array of targets, we did not identify any other significant differences in sensitivity between K-Ras wild-type and mutant cells, demonstrating the mechanistic relevance of this model to MEK inhibitors. Our data highlight the advantages of using this system in profiling molecularly targeted agents in order to identify potential mechanisms of resistance and in rationally identifying combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A70.

HGF Rescues Colorectal Cancer Cells from EGFR Inhibition via MET Activation

Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), is active in colorectal cancer (CRC). However, response rates range from only 10% to 20%. Here, we investigate hepatocyte growth factor (HGF)-dependent mesenchymal-epithelial transition factor (MET) activation as a mediator of cetuximab resistance through signal diversification in CRC cell lines. DiFi, GEO, and LIM1215 cells were treated with varying concentrations and combinations of EGF, HGF, cetuximab, and PHA-665752 (a highly specific MET kinase inhibitor). Biological end points included proliferation, cell cycle arrest, and apoptosis. Proliferation was measured using WST-1 assays and synergy investigated via isobolograms. Expression and signaling were examined using immunoblotting. EGFR and MET are coexpressed in these CRC cell lines, and dual receptor activation synergistically increased proliferation. Cetuximab inhibited cell growth by 60%-80% with an associated dephosphorylation of EGFR, MAPK, and/or AKT. Addition of HGF to cetuximab-treated cells phosphorylated MET, but not EGFR or ErbB3, restimulated the MAPK and AKT pathways, restored cell proliferation, and rescued cells from G1 arrest and apoptosis. Importantly, this effect could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating MET expression with RNAi. HGF-induced MET activation is a novel mechanism of cetuximab resistance in CRC. Inhibition of the HGF-MET pathway may improve response to EGFR inhibitors in CRC, and combination therapy should be further investigated.

Analysis of cellular phosphatidylinositol (3 ,4 ,5)-trisphosphate levels and distribution using confocal fluorescent microscopy

We have developed an immunocytochemistry method for the semiquantitative detection of phosphatidylinositol (3 ,4 ,5)-trisphosphate (PI(3,4,5)P3) at the cell plasma membrane. This protocol combines the use of a glutathione S-transferase-tagged pleckstrin homology (PH) domain of the general phosphoinositides-1 receptor (GST–GRP1PH) with fluorescence confocal microscopy and image segmentation using cell mask software analysis. This methodology allows the analysis of PI(3,4,5)P3 subcellular distribution in resting and epidermal growth factor (EGF)-stimulated HEK293T cells and in LIM1215 (wild-type phosphoinositide 3-kinase (PI3K)) and LIM2550 (H1047R mutation in PI3K catalytic domain) colonic carcinoma cells. Formation of PI(3,4,5)P3 was observed 5 min following EGF stimulation and resulted in an increase of the membrane/cytoplasm fluorescence ratio from 1.03 to 1.53 for HEK293T cells and from 2.2 to 3.3 for LIM1215 cells. Resting LIM2550 cells stained with GST–GRP1PH had an elevated membrane/cytoplasm fluorescence ratio of 9.8, suggesting constitutive PI3K activation. The increase in the membrane/cytoplasm fluorescent ratio was inhibited in a concentration-dependent manner by the PI3K inhibitor LY294002. This cellular confocal imaging assay can be used to directly assess the effects of PI3K mutations in cancer cell lines and to determine the potential specificity and effectiveness of PI3K inhibitors in cancer cells.

Abstract 705: Crosstalk with stromal fibroblasts contributes to EGFR inhibitor resistance in colorectal cancer cells

Abstract The mechanism for resistance to EGFR targeted therapies in colorectal cancer (CRC) with intact KRAS is still unknown. Host stromal cells contribute to tumorigenicity; however their effect on the sensitivity to EGFR targeted therapies has still to be determined. We hypothesized that crosstalk between stromal fibroblasts and CRC cancer cells contributes to cetuximab resistance via activation of the HGF/MET axis. Primary CRC associated fibroblasts (CAFs) were isolated and expanded from five surgical specimens. Fibroblasts were enriched by magnetic activated cell sorting (MACS®) and activated fibroblast lineage was confirmed by alpha smooth muscle actin (SMA) immunofluorescence. Supernatant HGF content and cellular mRNA expression levels in primary CRC CAFs, an embryonic lung fibroblast cell line (MRC-5), and a normal colon fibroblast cell line (CCD18) were determined by ELISA and quantitative RT-PCR. The effect of fibroblasts or HGF (75ng/ml) on the CRC cell lines LIM1215 and DiFi treated with cetuximab (5nM) for 72 hours was determined by co-culture using Transwells and WST-1 cell proliferation assays. Alterations in proliferation were compared by ANOVA. Mean fibroblast HGF secretion (8.3 ng/ml) and relative mRNA expression (2.0) could be augmented by co-culture with conditioned media (CM) from the CRC cell LIM1215 (14.1 ng/ml and 4.1, respectively) (Table). Further, co-culture of CRC cells with HGF, CCD18 cells, or primary CAFs reversed cetuximab-induced growth inhibition. LIM1215 proliferation was inhibited by cetuximab (55.3 ± 14.4%) which could be reversed by co-culture with either HGF (93.1 ± 10.2%) or CCD18 cells (107.2 ± 10.8%) (p<0.01 for both). Stromal-tumor interactions may play an important role in CRC resistance to cetuximab via the HGF/MET axis. Inhibition of both EGFR and MET may be necessary to realize the full potential of EGFR targeted therapies in CRC.Supernatant HGF levels and relative mRNA expression in fibroblasts treated with LIM1215 CM LIM1215 (Control)MRC5CCD18CAF3CAF4CAF5CAF6HGF (ng/ml) Ctrl<0.11.217.310.97.24.08.9LIM1215-CM<0.10.840.015.08.45.015.5 HGF mRNA Ctrl00.81.82.63.01.6 LIM1215-CM01.07.65.44.91.8 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 705.