EGFR-specific Tyrosine Kinase Inhibitors Research Articles

L858R emerges as a potential biomarker predicting response of lung cancer models to anti-EGFR antibodies: Comparison of osimertinib vs. cetuximab

EGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer.

Abstract 2532: Disrupting resistance to EGFR-specific tyrosine kinase inhibitors in TP53-mutated lung carcinoma cell lines by proteasome inhibition

Abstract Lung cancer is the leading cause of cancer related death. The most common form is non-small cell lung cancer (NSCLC). In a large proportion of NSCLC, driver mutations in protein kinases are found. One of the most frequently affected gene is the epidermal growth factor receptor (EGFR). Anti-cancer drugs have been developed that specifically inhibit mutated EGFR and are effective in anti-cancer therapy. However, resistance mediating mutations occur regularly during therapy limiting both treatment outcome and therapeutic options for follow up therapy. In sarcoma cell lines, resistance has already been overcome by a combination of proteasome inhibitors (PIs) with BH3 mimetics (Muenchow et al., 2020). In this case, drugs were combined that have an independent mechanism of action. In ALK-rearranged/TP53-mutated NSCLC cells, ALK-TKI resistance is also overcome by proteasome inhibition that mediates induction of the pro-apoptotic protein NOXA (Tanimoto et al., 2020). Since NOXA inhibits anti-apoptotic BCL-2 proteins, we hypothesize that combined treatment with receptor tyrosine kinase inhibitors (RTKIs) and PI or BCL-2 inhibitors (BCL2I) is an effective strategy for the treatment of EGFR mutated NSCLC tumors. To explore this hypothesis, we investigated cell death induction by combined administration of RTKI and PI in NSCLC cells differing in their mutational status, i.e., RTKI resistance. In addition, we analyzed the expression of effector BCL-2 family proteins (BAX, BAK, BOK) to determine their relevance to cell death induction and their informative value as predictive markers. By flow cytometric analysis of apoptosis markers (exposure of phosphatidyl serine, loss of mitochondrial membrane potential) we showed that the RTKI Osimertinib and PI Bortezomib synergistically induce cell death in H1650 and HCC4006 cells, independent of RTKI resistance. Importantly, Western blot analysis showed RTKI-induced accumulation of the pro-apoptotic BH3-only protein NOXA in the HCC4006 and H1650 cancer cell lines. Additionally, the H1975 showed a similar effect combining Osimertinib and ABT-199, a BCL2I, in flow cytometric analysis and Western blot. In summary, the presented experiments support the hypothesized synergistic cell death induction by RTK-inhibitors and proteasome inhibition as well as BCL-2-inhibition. Studying the molecular mechanism of synergistic efficacy of RTKI/PI or RTKI/BCL2I combination therapies may form the basis for a general therapeutic concept which relies on simultaneous blocking of driver mutations and survival-promoting BCL-2 proteins. Citation Format: Franziska Orth, Benjamin Schaefer, Annika Harsch, Sandra Weller, Tobias Beigl, Lynn Barber, Hans-Georg Kopp, Frank Essmann. Disrupting resistance to EGFR-specific tyrosine kinase inhibitors in TP53-mutated lung carcinoma cell lines by proteasome inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2532.

The Co-expression of HER Family Members and CD109 is common in Pancreatic Cancer.

In the absence of biomarkers for the early detection of pancreatic cancer and the development of more effective therapeutic interventions, pancreatic cancer is projected to become the second leading cause of death from cancer in the Western world. In the past four decades, aberrant expression and activation of human epidermal growth factor receptor (EGFR, also called HER) family members have been reported in a wide range of human cancers. Currently, of the various types of drugs targeting one or more members of the HER family, only the EGFR-specific tyrosine kinase inhibitor erlotinib and more recently Zenocutuzumab, a HER-2/HER3 bispecific antibody, were granted breakthrough therapy designation by the FDA for the treatment of patients with pancreatic cancer. However, the therapeutic benefit may be modest in some patients. Hence there is an urgent need for the identification of biomarkers as prognostic indicators, therapeutic targets, and for the response to therapy and the development of more effective therapeutic agents for this highly heterogeneous cancer. In this study, for the first time, we investigated the relative expression and prognostic significance of all members of the HER family, the type-III EGFR mutant (EGFRvIII), and CD109 in tissue microarrays (TMAs) and whole tumour specimens (WTS) from pancreatic cancer patients by immunohistochemistry. We found the positive expression of wild-type EGFR (wt-EGFR) (63%, 4.7%), HER2 (75%, 14%), HER3 (none, 14%), HER4 (45%, 21%), EGFRvIII (5%, 3%), and CD109 (67%, 55%) in TMAs (USA) and WTS (UK) from pancreatic cancer patients respectively. Our results also show that the co-expression of HER family members with CD109 occurs frequently in patients with pancreatic cancer. In addition, the co-expression of HER4/CD109 may also be associated with poorer overall survival in such patients. In this article, we shall discuss these findings and their implications and future opportunities for more effective targeting of HER positive pancreatic cancer using the HER inhibitors in combination with other drugs and therapeutic interventions.

EGFR exon 20 insertion variants A763_Y764insFQEA and D770delinsGY confer favorable sensitivity to currently approved EGFR-specific tyrosine kinase inhibitors

Background: The EGFR exon 20 insertions (ex20ins) D770_N771insSVD and V769_D770insASV are most frequent in non-small-cell lung cancer (NSCLC) and are associated with intrinsic resistance to currently approved EGFR tyrosine kinase inhibitors (TKIs). A763_Y764insFQEA and D770delinsGY, respectively, account for 3%-8% and 2.0%-4.8% of EGFR ex20ins in NSCLC and are associated with a more favorable response to EGFR-specific TKIs as per case reports. The aim of this study was to elucidate the molecular structures of these mutants and their binding affinities to diverse EGFR TKIs and compare the clinical outcomes in NSCLC patients harboring these mutations. Methods: A real-world cohort study was conducted to evaluate and compare the clinical outcomes of EGFR TKIs among NSCLC patients with different EGFR ex20ins mutants in response to EGFR TKIs. The structures of A763_Y764insFQEA and D770delinsGY were also analyzed and drug binding simulations were performed. Results: With a median follow-up of 24.0months, the first-line objective response rate (ORR), disease control rate (DCR), and median progression-free survival (PFS) were, respectively, 0 (0/16), 50.0% (8/16), and 2.07months (95%CI, 0-6.25) in patients harboring D770_N771insSVD and V769_D770insASV variants and 33.3% (4/12), 83.3% (10/12), and 9.97months (95%CI, 4.75-15.19) in patients with A763_Y764insFQEA and D770delinsGY variants. There was a significant difference between the PFS of these two subgroups (median, 9.97 vs.2.07months, HR = 0.33, 95%CI, 0.13-0.85, p = 0.02). Similarly, the PFS was significantly longer after second-line treatment with EGFR TKIs in patients harboring A763_Y764insFQEA and D770delinsGY compared to those with other insertions (median, 6.77 vs.2.23months, HR = 0.14, p < 0.001). Computational simulations indicated that A763_Y764insFQEA and D770delinsGY mutants were structurally similar to wild-type EGFR. In contrast, the C-helix and phosphate-binding loop of D770_N771insSVD and V769_D770insASV had shifted into the drug-binding pocket, resulting in significant steric hindrance and a lack of affinity for the currently approved EGFR inhibitors. Conclusion: NSCLC patients harboring A763_Y764insFQEA and D770delinsGY insertions of EGFR are responsive to the currently approved EGFR TKIs as opposed to patients with the D770_N771insSVD and V769_D770insASV variants. Therefore, A763_Y764insFQEA and D770delinsGY should be classified as active mutations among heterogeneous EGFR ex20ins subtypes and the carriers can be treated with the suitable EGFR TKIs.

EP08.02-172 Effect of EGFR Inhibition on Bone Health in NSCLC Patients without Skeletal Metastasis

The Epidermal Growth Factor Receptor (EGFR) signaling has known effects in bone homeostasis by modulating proliferation and differentiation of osteoblasts, osteoclasts and bone marrow osteoprogenitor cells. Studies in EGFR-deficient mice or in mice treated with EGFR-inhibitors, showed impaired bone formation. Patients affected by metastatic non-small cell lung cancer (NSCLC) with sensitizing mutations of the EGFR gene (mEGFR) are treated with EGFR-specific tyrosine kinase inhibitors (EGFR-TKIs), obtaining long-term disease control and improvement of quality of life (QOL).

Abstract LB517A: The role of EGFR in resistance to tucatinib and its therapeutic implications

Abstract Tucatinib (Tuc) was recently approved for metastatic disease and is moving towards the early setting in HER2+ breast cancer (BC). Given the increasing clinical use of Tuc, resistance will likely soon emerge as a challenge. Here, we explore the yet unknown mechanisms of resistance to Tuc and identify treatment strategies to overcome it. Our recently developed models of BT474 (AZ and ATCC) with acquired resistance to Tuc (TucR) and their sensitive parental (P) were used. DNA-seq, RNA-seq, and RPPA/western blot were performed. Knockdown studies were performed using EGFR siRNA. Drug efficacy studies involved cell growth assays by imaging-based or methylene blue assays. We recently reported (SABCS 2021) that our BT474 TucR models acquired EGFR amplification. The TucR cells displayed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6, which were substantially suppressed by the EGFR-specific tyrosine kinase inhibitor (TKI) gefitinib (Gef) or even further when combined with Tuc. Our new results demonstrate that EGFR knockdown selectively inhibits the growth and pHER2 levels in TucR vs P cells, supporting our hypothesis that heterodimerization of amplified EGFR with HER2 leads to higher pHER2 levels in TucR cells. We have recently also shown that TucR models were hypersensitive to Gef and this inhibition was further enhanced with Gef+Tuc, implying their survival dependence on EGFR. Here, we demonstrate that the TucR cells made resistant to 200nM Tuc maintain their resistant growth and elevated EGFR-dependent signaling even when exposed to 500nM, and can begrown as xenografts in the presence of clinically relevant dose of Tuc, emphasizing their true resistance via amplified EGFR. Importantly, both TucR models vs P cells were cross-resistant to trastuzumab but maintain partial sensitivity to TDM1. While the EGFR-specific antibody cetuximab (Cet) was partially effective as a single agent only in the ATCC model, it potently inhibited growth and induced cell killing in combination with Tuc in both models. A significantly greater inhibition in cell growth and survival was also observed when trastuzumab or TDM1 was combined with either Gef or Cet. Taken together, our results suggest that the activation of HER2 and the resistant growth and survival in the TucR models is completely dependent on the amplified EGFR, which we are currently further corroborating by additional mechanistic and xenograft studies. Whilst we have previously reported that resistance to lapatinib and neratinib confer cross-resistance to Tuc, our recent findings show that resistance to Tuc may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Overall, our novel findings hold crucial implications in light of the current treatment landscape of HER2+ BC and biomarkers of resistance, and places a particular emphasis on considerations to sequence currently available TKIs. Citation Format: Jamunarani Veeraraghavan, Fu-Tien Liao, Tia Gordon, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Yingjie Zhu, Juber A. Patel, Andrea Gazzo, Fabio Stossi, Michael A. Mancini, Carolina Gutierrez, Britta Weigelt, Jorge S. Reis-Filho, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. The role of EGFR in resistance to tucatinib and its therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB517A.

Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models.

Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anti‐cancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first‐, second‐, and third‐generation drugs. To explore alternatives to such whack‐a‐mole strategies, we simulated in patient‐derived xenograft models the situation of patients receiving first‐line TKIs. Monotherapies comprising approved first‐line TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drug–drug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistance‐conferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing next‐generation TKIs.

EGFR-Specific Tyrosine Kinase Inhibitor Modifies NK Cell-Mediated Antitumoral Activity against Ovarian Cancer Cells.

The adverse prognosis of most patients with ovarian cancer is related to recurrent disease caused by resistance to chemotherapeutic and targeted therapeutics. Besides their direct activity against tumor cells, monoclonal antibodies and tyrosine kinase inhibitors (TKIs) also influence the antitumoral activity of immune cells, which has important implications for the design of immunotherapies. In this preclinical study, we treated different ovarian cancer cell lines with anti-epidermal growth factor receptor (EGFR) TKIs and co-incubated them with natural killer (NK) cells. We studied treatment-related structural and functional changes on tumor and immune cells in the presence of the anti-EGFR antibody cetuximab and investigated NK-mediated antitumoral activity. We show that long-term exposure of ovarian cancer cells to TKIs leads to reduced responsiveness of intrinsically sensitive cancer cells over time. Inversely, neither long-term treatment with TKIs nor cetuximab could overcome the intrinsic resistance of certain ovarian cancer cells to anti-EGFR agents. Remarkably, tumor cells pretreated with anti-EGFR TKIs showed increased sensitivity towards NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In contrast, the cytokine secretion of NK cells was reduced by TKI sensitization. Our data suggest that sensitization of tumor cells by anti-EGFR TKIs differentially modulates interactions with NK cells. These data have important implications for the design of chemo-immuno combination therapies in this tumor entity.

Abstract 2203: A SRM/MRM based targeted proteomics strategy for absolute quantification of potential biomarkers of TKI sensitivity in EGFR mutated lung adenocarcinoma

Abstract Lung cancer remains the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor(EGFR) is the primary driver oncogene in 10-30% lung adenocarcinoma. Kinase domain mutations in EGFR (L858R and E746-A750 deletion) provide sensitivity to EGFR-specific tyrosine kinase inhibitors (TKIs). Despite good tumor inhibitory effects, after approximately a year, patients acquire resistance to EGFR TKIs. We have carried out a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) on a Thermo Orbitrap Elite to identify and characterize the dynamics of tyrosine phosphorylation upon TKI treatment of human lung adenocarcinoma cell lines. The degree of inhibition of phosphorylation of several phospho-sites correlated with the extent of TKI-sensitivity. Several kinases and adaptor proteins that were significantly differentially phosphorylated include AHNAK-Y160, -Y715, STAT5A-Y694, DAPP1-Y139, ERRFI1-Y394, NEDD9-Y164, NF1-Y2579, INNPL1-Y1135, suggesting that these are a subset of potential biomarkers of TKI response. To corroborate our results, the next logical step is to carry out clinical validation of these phospho-sites in human lung adenocarcinoma patients, however no absolute quantification assay is currently available for these tyrosine phosphorylated peptides in human samples. The MS method most often used to complement shotgun methods is selected/multiple reaction monitoring (SRM/MRM) because of its high sensitivity, speed and dynamic range. We have performed SRM/MRM-MS using a nano-flow high performance liquid chromatography instrument coupled to a nano-electrospray ionization source attached to a Triple Quadrupole MS (Agilent 6495). In this method, digested proteins are chromatographically separated and ionized. The resulting precursor ions are m/z filtered in the first quadrupole(Q1) and collision induced dissociation is carried out in Q2 and the resultant product ions are m/z filtered in Q3. Each precursor-product pair is termed as a transition. For absolute quantification, heavy isotope labelled versions of the target peptides are spiked in biological samples to build a calibration curve. For the robust assay development, we have carried out the initial empirical refinement process using the heavy labelled versions of the target peptides, including selection of optimal transitions, normalizing retention times of peptides, running peptide stability assays and digestion time course experiments. We are currently validating these assays in human lung adenocarcinoma cell lines with known EGFR mutations with or without treatment with 1st and 3rd generation EGFR TKIs. The overall goal of these targeted studies is to develop absolute quantitation assays to precisely and accurately quantify these potential biomarkers in clinical samples. Citation Format: Shivangi Awasthi, Xu Zhang, Tapan Maity, Benjamin L. Oyler, David R. Goodlett, Udayan Guha. A SRM/MRM based targeted proteomics strategy for absolute quantification of potential biomarkers of TKI sensitivity in EGFR mutated lung adenocarcinoma [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 2203. doi:10.1158/1538-7445.AM2017-2203

Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration.

A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis.

Research Progress of EGFR-TKI Therapy for Patients with Central Nervous System Metastases from Non-small Cell Lung Cancer

约50%的非小细胞肺癌会出现中枢神经系统转移，从而导致不良预后。非小细胞肺癌患者中存在表皮生长因子受体（epidermal growth factor receptor, EGFR）突变，这部分患者对EGFR酪氨酸激酶抑制剂（EGFRtyrosine kinase inhibitors, EGFR-TKI）的治疗显示出了良好的耐受性及疗效。EGFR-TKI对非小细胞肺癌中枢神经系统转移也显示出了一定的疗效。本文针对EGFR-TKI药物对于非小细胞肺癌中枢神经系统转移的治疗进展进行综述。

Local Epidermal Growth Factor Receptor Signaling Mediates the Systemic Pathogenic Effects of Staphylococcus aureus Toxic Shock Syndrome.

Secreted factors of Staphylococcus aureus can activate host signaling from the epidermal growth factor receptor (EGFR). The superantigen toxic shock syndrome toxin-1 (TSST-1) contributes to mucosal cytokine production through a disintegrin and metalloproteinase (ADAM)-mediated shedding of EGFR ligands and subsequent EGFR activation. The secreted hemolysin, α-toxin, can also induce EGFR signaling and directly interacts with ADAM10, a sheddase of EGFR ligands. The current work explores the role of EGFR signaling in menstrual toxic shock syndrome (mTSS), a disease mediated by TSST-1. The data presented show that TSST-1 and α-toxin induce ADAM- and EGFR-dependent cytokine production from human vaginal epithelial cells. TSST-1 and α-toxin also induce cytokine production from an ex vivo porcine vaginal mucosa (PVM) model. EGFR signaling is responsible for the majority of IL-8 production from PVM in response to secreted toxins and live S. aureus. Finally, data are presented demonstrating that inhibition of EGFR signaling with the EGFR-specific tyrosine kinase inhibitor AG1478 significantly increases survival in a rabbit model of mTSS. These data indicate that EGFR signaling is critical for progression of an S. aureus exotoxin-mediated disease and may represent an attractive host target for therapeutics.

Activity of AUY922 in NSCLC patients with EGFR exon 20 insertions.

8015 Background: EGFR exon 20 insertions (ins20) represent a rare subtype (4%) of EGFR mutations and are refractory to EGFR-specific tyrosine kinase inhibitors (TKIs). No effective targeted therapi...

An Inducible TGF-β2-TGFβR Pathway Modulates the Sensitivity of HNSCC Cells to Tyrosine Kinase Inhibitors Targeting Dominant Receptor Tyrosine Kinases.

The epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of head and neck squamous cell carcinomas (HNSCC), and molecularly targeted therapy against the EGFR with the monoclonal antibody cetuximab modestly increases overall survival in head and neck cancer patients. We hypothesize that co-signaling through additional pathways limits the efficacy of cetuximab and EGFR-specific tyrosine kinase inhibitors (TKIs) in the clinical treatment of HNSCC. Analysis of gene expression changes in HNSCC cell lines treated 4 days with TKIs targeting EGFR and/or fibroblast growth factor receptors (FGFRs) identified transforming growth factor beta 2 (TGF-β2) induction in the three cell lines tested. Measurement of TGF-β2 mRNA validated this observation and extended it to additional cell lines. Moreover, TGF-β2 mRNA was increased in primary patient HNSCC xenografts treated for 4 weeks with cetuximab, demonstrating in vivo relevance of these findings. Functional genomics analyses with shRNA libraries identified TGF-β2 and TGF-β receptors (TGFβRs) as synthetic lethal genes in the context of TKI treatment. Further, direct RNAi-mediated silencing of TGF-β2 inhibited cell growth, both alone and in combination with TKIs. Also, a pharmacological TGFβRI inhibitor similarly inhibited basal growth and enhanced TKI efficacy. In summary, the studies support a TGF-β2-TGFβR pathway as a TKI-inducible growth pathway in HNSCC that limits efficacy of EGFR-specific inhibitors.

Abstract A66: Colonization of human ovarian cancer cells is promoted by cancer-fibroblast interaction via TGFα-EGFR signaling

Abstract Tumor colonization involves reciprocal interactions between the metastatic cancer cells and various stromal cells in foreign microenvironment. Fibroblasts are the second most numerous cell types in omentum, which is the commonest site of ovarian cancer metastasis. However, the crosstalk between metastatic ovarian cancer cells and fibroblasts that involved in tumor colonization remains largely unknown. In this study, we used a three-dimensional (3D) co-culture model to study the role of normal fibroblasts on tumor colonization of ovarian cancer cells. Human ovarian cancer cell lines, which were established from peritoneal ascites, were seeded on normal fibroblasts embedded in extracellular matrix (Matrigel). Our results showed that ovarian cancer spheroids formed in Matrigel with fibroblasts were significantly larger and also more invasive than those formed in Matrigel without fibroblasts, suggesting the cancer-fibroblast interaction promotes the colonization of ovarian cancer cells. To search the signal, we compared the cytokine profile of the 3D co-culture with 3D monoculture of ovarian cancer cells/fibroblasts. De novo production of TGF-α was found in the fibroblasts of the co-culture. To determine whether TGF-α promotes the colonization of ovarian cancer cells, we treated the monoculture of ovarian cancer cells in Matrigel with recombinant human TGF-α (rhTGF-α). rhTGF-α significantly promoted the growth and invasion of ovarian cancer spheroids. Additionally, our qRT-PCR results demonstrated that EGFR, a receptor for TGF-α, was over-expressed in primary human ovarian cancer tissues when compared to non-malignant human ovarian tissues. To test whether fibroblast-derived TGF-α promotes tumor colonization of ovarian cancer cells through EGFR, we treated our in-vitro colonization assays with an EGFR inhibitor, AG1478. AG1478 significantly reduced the growth and invasion of rTGFα-induced ovarian cancer spheroids and the cancer spheroids formed in the co-culture, indicating the colonization of human ovarian cancer cells is promoted by cancer-fibroblast interaction via TGFα-EGFR signaling. We also found that Erlotinib and Gefitinb, two clinically approved EGFR-specific tyrosine kinase inhibitors, significantly reduced the growth and invasion of ovarian cancer spheroids in our in-vitro colonization assays. This result implicates the peritoneal metastasis of ovarian cancer can be potentially treated by EGFR-targeted therapy. (This project is supported by CUHK Research Committee Funding). Citation Format: Tat-San Lau, Tony Kwok-Hung Chung, Tak-Hong Cheung, So-Fan Yim, Chi-Hang Wong, Joseph Kwong. Colonization of human ovarian cancer cells is promoted by cancer-fibroblast interaction via TGFα-EGFR signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A66.

Abstract 5116: The calcium channel subunit CACNG4 plays a role in breast cancer metastasis.

Abstract Introduction: Previously, we used aCGH to analyze genomic alterations in primary breast tumors with and without lymph node (LN) metastasis. A significant correlation was found between gain of chromosome 17q24 and LN metastasis. Selected genes including L-type calcium channel voltage dependent gamma subunit 4 (CACNG4) were further validated for increased protein expression in invasive breast tumors. CACNG4 is involved in calcium channel regulation in excitable cells of muscles and the brain. It is the only gamma subunit that also shows expression in non-excitable cells, such as glial and intestinal epithelial cells, suggestive of additional functions. It is over-expressed in glioblastoma multiforme cell lines resistant to EGFR inhibitors. Breast cancer gene expression datasets (NKI and IPC) identified an association of CACNG4 expression with LN metastasis and ER positivity. In the present study, we characterized CACNG4 in breast cancer with particular attention to its metastasis-related ability. Methodology: RNA interference was used for knockdown studies in cell lines followed by cell migration, invasion, aggregation and adhesion assays. Intracellular calcium influx in response to calcium channel agonists and antagonists was measured using the Fluo-4 calcium indicator dye. Results: CACNG4 showed variable expression across a panel of breast cancer cell lines. MCF10A (a non malignant cell line) does not express CACNG4, while it is highly expressed in ER positive cell lines (MCF7, MDA-MB-361, CAMA1) and lower in EGFR high cell lines (MDA-MB-468 and MDA-MB-231). CACNG4 knockdown inhibited cell migration and invasion in MCF7 and MDA-MB-231 cells. Conversely, cell adhesion and cell-cell aggregation was increased. RT-PCR revealed an inverse relation between CACNG4 expression and PKC-zeta specifically, which is involved in tight junction formation and shows low expression in breast, prostate and renal cancers. Thus, CACNG4 may play a role in de-adhesion and cell dissemination during metastasis. Blocking calcium channels with amlopidine had an anti-proliferative affect. Serum stimulation of EGFR high cell lines resulted in calcium influx, that was blocked in a dosage dependent manner with amlodipine, and synergistically blocked further with the EGFR specific tyrosine kinase inhibitor tryphostin AG1478. Knockdown of CACNG4 resulted in increased calcium influx suggesting a role in keeping the channel closed. Cells also showed resistance to amlodipine, requiring higher doses to block calcium influx. Conclusions: L-type channels are active in non-excitable breast cancer cells, and are affected by EGFR activity. CACNG4 maintains these channels in a closed state, modulating calcium influx, and inhibiting PKC-zeta expression which may result in metastatic function. The ability of calcium channel blockers to target channels in a CACNG4 dependent manner suggests novel treatment possibilities for invasive breast cancer. Citation Format: Nisha Kanwar, Ranju Nair, Dong-Yu Wang, Susan J. Done. The calcium channel subunit CACNG4 plays a role in breast cancer metastasis. [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 5116. doi:10.1158/1538-7445.AM2013-5116

Abstract 3387: Tyrosine kinase inhibitors induce TGF-β2 expression in head and neck squamous cell carcinoma cell lines as a mechanism of acquired resistance.

Abstract Worldwide, head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer. Because the epidermal growth factor receptor (EGFR) is overexpressed in about 90% of HNSCC tumors, molecularly targeted therapy against EGFR was thought to hold promise for HNSCC treatment. Cetuximab, a monoclonal antibody against EGFR, has been approved to treat HNSCC in the US. Still the 5-year survival rate for HNSCC is only 40-50%, despite the use of cetuximab in the clinic. The modest effect of cetuximab on survival indicates that EGFR inhibition alone will not be effective in treating HNSCC. The efficacy of cetuximab in treating HNSCC may be limited by acquired resistance or the activity of alternative dominant growth factor pathways in HNSCC. Previously, our lab has shown that in addition to EGFR signaling, the fibroblast growth factor 2 (FGF2) and its receptors (FGFRs) participate as an important autocrine signaling pathway in some gefitinib-insensitive HNSCC cell lines, further indicating that inhibiting the EGFR pathway alone will not be effective in treating HNSCC. To identify genes that change in response to EGFR-specific tyrosine kinase inhibitors (TKIs) and to FGFR-specific TKIs that may mediate acquired resistance, we performed an Affymetrix GeneChip screen in which the cell lines UMSCC25, 584-A2, and Ca9-22 were treated for 4 days with 0.3μM AZ8010, a TKI selective for FGFRs, and/or 0.1μM gefitinib, an EGFR-selective TKI. We found that in all three cell lines, TGF-β2 mRNA was upregulated following blockade of the dominant receptor pathway. Both ELISA and qRT-PCR were used to validate this induction of TGF-β2 in these three cell lines. Treatment with a small molecule inhibitor of TGF-β receptor I (TGFβRI) provided an additive reduction of clonogenic growth when combined with EGFR and/or FGFR TKIs. Additionally, silencing TGF-β2 with shRNA in UMSCC25 cells lead to an additive decrease in clonogenic growth in combination with EGFR and/or FGFR TKIs. Furthermore, we observed an induction of NF-κB activity in the cell lines following treatment with EGFR and/or FGFR TKIs. This induction was mitigated by the use of a TGF-β2 neutralizing antibody, suggesting that TGF-β2 is signaling through a non-canonical pathway in our model. In addition to observing a rapid increase in TGF-β2 expression, the cell line UMSCC25 was chronically adapted to increasing concentrations of gefitinib over the course of several months. We observed a sustained increase in TGF-β2 expression in the resistant cells compared to the control cells. This data suggests that TGF-β2 induction may provide a novel mechanism of acquired resistance to TKIs, and supports the hypothesis that combination therapy will be more effective than monotherapy in treating HNSCC. Citation Format: Emily K. Kleczko, Lydia R. Heasley, Marianne E. Marshall, Katherine R. Singleton, Lynn E. Heasley. Tyrosine kinase inhibitors induce TGF-β2 expression in head and neck squamous cell carcinoma cell lines as a mechanism of acquired resistance. [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 3387. doi:10.1158/1538-7445.AM2013-3387

A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop

Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M ‘gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.

Dacomitinib, an Irreversible Pan-ErbB Inhibitor Significantly Abrogates Growth in Head and Neck Cancer Models That Exhibit Low Response to Cetuximab

Aberrant epidermal growth factor (EGF) signaling is associated with tumor growth in squamous cell carcinoma of the head and neck in humans (HNSCC), and is a major focus of targeted therapy. Cetuximab, a monoclonal antibody against EGFR, has been successful at prolonging survival but has only a 10% tumor shrinkage response rate in a clinical setting. The goal of this study was to compare dacomitinib (PF-00299804), a next generation small molecule tyrosine kinase inhibitor that irreversibly blocks multiple HER family receptors (HER-1 (EGFR), HER-2 and HER-4 tyrosine kinases), to cetuximab, the current FDA approved anti-EGFR medication for HNSCC and erlotinib, an EGFR specific small molecule tyrosine kinase inhibitor. Dacomitinib, erlotinib and cetuximab were tested in a panel of 27 HNSCC cell lines. Treatment with 100 ug/ml of cetuximab or 1 uM of erlotinib inhibited growth by at least 50% in 7/27 cell lines, while treatment with 1 uM of dacomitinib had similar growth inhibition in 17/27 lines. Cell lines representing three levels of sensitivity to dacomitinib were further examined using Western blots, cell cycle and apoptosis analysis. Treatment with 100 nM of dacomitinib reduced EGFR activity and downstream AKT and ERK pathways more effectively than treatment with 100 ug/ml of cetuximab in all ten tested lines. Although both compounds induced apoptosis at similar levels, dacomitinib caused greater G0/G1 arrest. Sensitivity to EGFR blockade was associated with levels of EGFR and ERK and was not associated with common oncogenic mutations and copy number variations. Phosphorylated and total EGFR and ERK levels correlate with sensitivity to both cetuximab and dacomitinib. Three of the four lines in the exquisitely sensitive group had the highest levels of phosphorylated and total EGFR and ERK among the ten lines selected, while the three resistant lines collectively had the lowest levels. Neither pAKT nor tAKT was associated with sensitivity.

Incidence and patterns of ALK FISH abnormalities seen in a large unselected series of lung carcinomas

BackgroundAnaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements have been reported in 2-13% of patients with non-small cell lung cancer (NSCLC). Patients with ALK rearrangements do not respond to EGFR-specific tyrosine kinase inhibitors (TKIs); however, they do benefit from small molecule inhibitors targeting ALK.ResultsIn this study, fluorescence in situ hybridization (FISH) using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in a large unselected cohort of 1387 patients with a referred diagnosis of non-small cell lung cancer (1011 of these patients had a histologic diagnosis of adenocarcinoma). The abnormal FISH signal patterns varied from a single split signal to complex patterns. Among 49 abnormal samples (49/1387, 3.5%), 32 had 1 to 3 split signals. Fifteen samples had deletions of the green 5′ end of the ALK signal, and 1 of these 15 samples showed amplification of the orange 3′ end of the ALK signal. Two patients showed a deletion of the 3′ALK signal. Thirty eight of these 49 samples (38/1011, 3.7%) were among the 1011 patients with confirmed adenocarcinoma. Five of 8 patients with ALK rearrangements detected by FISH were confirmed to have EML4-ALK fusions by multiplex RT-PCR. Among the 45 ALK-rearranged samples tested, only 1 EGFR mutation (T790M) was detected. Two KRAS mutations were detected among 24 ALK-rearranged samples tested.ConclusionsIn a large unselected series, the frequency of ALK gene rearrangement detected by FISH was approximately 3.5% of lung carcinoma, and 3.7% of patients with lung adenocarcinoma, with variant signal patterns frequently detected. Rare cases with coexisting KRAS and EGFR mutations were seen.