Protein Expression Of p-EGFR Research Articles

Loss of p16 expression is a risk factor for recurrence in sinonasal inverted papilloma.

The purpose of this study was to evaluate p16, p53, EGFR, pEGFR protein expression and HPV infection as possible markers of tumor progression in a series of sinonasal inverted papilloma (SNIP) and sinonasal squamous cell carcinoma (SNSCC). A series of 49 SNIP, 11 SNSCC associated with SNIP (SNIP-SNSCC) and 52 SNSCC not associated with SNIP were analyzed for p16, p53, EGFR, and phosphorylated EGFR (pEGFR) expression by immunohistochemistry. Human papillomavirus (HPV) infection status was evaluated by DNA-PCR. Results were correlated to clinical and follow-up data. Reduced or loss of p16 expression was observed in 18% SNIP, 64% SNIP-SNSCC and 87% of SNSCC. Reduced or loss p16 staining in SNIP correlated with shorter recurrent SNIP-free follow-up. In contrast, p16 expression was not predictive of recurrent SNSCC in cases with SNIP-SNSCC and SNSCC. P53, EGFR, and pEGFR expression did not differ between the tumor groups, nor were they related to recurrent SNIP-free follow-up or recurrent SNSCC. Oncogenic HPV types 16 and 18 were detected in 5% of SNIP and 18% of SNIP-SNSCC, but not in SNSCC. There was no correlation between HPV infection and >70% p16 immunostaining. HPV infection appears to play a minor role in SNIP and SNSCC and p16 immunostaining does not appear a valid surrogate marker for HPV. However, reduced or loss p16 expression may have prognostic value as a risk marker for recurrent SNIP.

Anti-proliferative tirucallane triterpenoids from gum resin of Boswellia sacra

Eight new tirucallane triterpenoids (1–2, 5–10) along with two known compounds (3–4) were isolated from the gum resin of Boswellia sacra. Their structures were elucidated by extensive physicochemical and spectroscopic analysis, as well as computational calculations, and single crystal X-ray diffraction. Spirosacraoic acid A (1) and B (2), possess an unusual 6/5/6/5 rearranged spirocyclic carbon skeleton. All the isolates were evaluated for their anti-proliferative activity against two tumor cell lines (HepG2 and HCT-116 cells). Compound 10 displayed remarkable inhibitory activity against HepG2 cells in a dose-dependent manner with the IC50 value of 28.01 μM. High content analysis (HCA) showed that 10 induces apoptosis in HepG2 cells. The western blotting results revealed that 10 could up-regulate the ratio of the expression of Bax/BCL-2, and promote the caspase 3 activation and PARP cleavage. Mechanically, molecular modeling studies demonstrated that 10 could dock into EGFR active site. Meanwhile 10 significantly decreased the protein expression of p-EGFR. Furthermore, inhibition of EGFR by addition of EGFR siRNA enhanced the growth inhibitory effects of 10 on HepG2 cells, indicating that the anti-tumor effect of 10 on HepG2 cells was mediated by inhibition of EGFR.

Functional and mutational analysis after radiation and cetuximab treatment on prostate carcinoma cell line DU145

BackgroundEpidermal Growth Factor Receptor is often overexpressed in advanced prostate carcinoma. In-vitro-studies in prostate carcinoma cell line DU145 have demonstrated increased sensibility to radiation after cetuximab treatment, but clinical data are not sufficient to date.MethodsWe analyzed effects of radiation and cetuximab in DU145 and A431 using proliferation, colony-forming-unit- and annexin-V-apoptosis-assays. Changes in protein expression of pEGFR and pERK1/2 after radiation and cetuximab treatment were analyzed. Using NGS we also investigated the impact of cetuximab long-term treatment.ResultsCell counts in DU145 were reduced by 44% after 4 Gy (p = 0.006) and 55% after 4 Gy and cetuximab (p < 0.001). The surviving fraction (SF) was 0.69 after 2 Gy, 0.41 after 4 Gy and 0.15 after 6 Gy (each p < 0.001). Cetuximab treatment did not alter significantly growth reduction in 4 Gy radiated DU145 cells, p > 0.05 or SF, p > 0.05, but minor effects on apoptotic cell fraction in DU145 were detected. Using western blot, there were no detectable pEGFR and pERK1/2 protein signals after cetuximab treatment. No RAS mutation or HER2 amplification was detected, however a TP53 gen-mutation c.820G > T was found.ConclusionsRadiation inhibits cell-proliferation and colony-growth and induces apoptosis in DU145. Despite blocking MAP-Kinase-pathway using cetuximab, no significant radiation-sensitizing-effect was detected. Cetuximab treatment did not induce resistance-mutations. Further research must clarify which combination of anti-EGFR treatment strategies can increase radiation-sensitizing-effects.

Human Neutrophil Elastase Mediates MUC5AC Hypersecretion via the Tumour Necrosis Factor-α Converting Enzyme-Epidermal Growth Factor Receptor Signalling Pathway in vivo

Objectives: The objective of this study is to examine the role of the tumour necrosis factor-α converting enzyme-epidermal growth factor receptor (TACE-EGFR) pathway in human neutrophil elastase (HNE)-induced MUC5AC mucin expression in mice. Method: Four groups of mice, treated with HNE alone (HNE group), HNE plus TACE inhibitor (HNE + TAPI-2 group), HNE plus EGFR inhibitor (HNE + AG1478 group), and untreated (control group), were used in the experiment. Histopathological changes were monitored by haematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. TACE, EGFR, and MUC5AC expression in the nasal mucosa were determined using immunohistochemistry. The expression of p-EGFR, EGFR, and TACE protein was analysed on Western blots, and MUC5AC protein levels were assessed via ELISA. TACE, EGFR, and MUC5AC expression in the nasal mucosa were determined using real-time quantitative PCR. Results: Compared to the control group, HE-stained tissues from the HNE group showed an irregular epithelium as well as goblet cell and submucosal glandular hyperplasia. In the nasal mucosa, strongly positive fuchsia granules were seen in PAS staining and significant increases in TACE, EGFR, MUC5AC mRNA, and protein expression were detected (p < 0.01). The HNE + TAPI-2 and HNE + AG1478 groups had significantly less goblet cell and submucosal gland hyperplasia as well as weaker PAS staining. Compared to mice treated with HNE alone, in HNE + TAPI-2-treated mice, the levels of TACE, EGFR, and MUC5AC mRNA and protein as well as p-EGFR protein were significantly reduced (p < 0.01). In HNE + AG1478-treated mice, EGFR and MUC5AC mRNA and protein levels and p-EGFR protein expression were reduced significantly (p < 0.01), but the difference in TACE mRNA and protein expression between the HNE + AG1478 and HNE groups was not significant (p > 0.05). Conclusion: Using a newly developed, stable experimental model of nasal hypersecretion in mice, we showed that TAPI-2 or AG1478 inhibited HNE-induced MUC5AC production. This suggests that MUC5AC mucin expression in vivo is mediated by a cascade involving the HNE-TACE-EGFR signalling pathway.

Abstract 3325: EGFR mutation and protein expression analysis in sinonasal inverted papilloma and squamous cell carcinoma

Abstract Introduction Inverted sinonasal papilloma has been demonstrated to be precursor to a subset of sinonasal squamous cell carcinoma and to carry frequent mutations in EGFR exon 20. The aim of this study was to evaluate EGFR mutation and protein expression as risk marker for malignant transformation of inverted papillomas. Experimental procedures The total number of samples studied was 41 inverted papillomas (ISP) and 32 squamous carcinomas (SCC). We defined patients with one single ISP, with multiple ISP (ISP-ISP) or ISP with malignant transformation (ISP-transformed). In addition, we classified SCC related with ISP (SCC-ISP) and those without relation (SCC de novo). EGFR exon 20 was amplified by PCR and analyzed by Sanger sequencing using the ABI PRISM 3100 and 3730 Genetic Analyzer, (Applied Biosystems, Foster City CA). Immunohistochemistry was performed on an automatic staining workstation (Dako Autostainer Plus; DakoCytomation, Glostrup, Denmark) using the antibody anti-pEGFR clone D7A5 (Cell Signaling Technology, Cambridge, UK). Results were evaluated by two experienced investigators (BV and MM). Results We found EGFR exon 20 mutations in 62% (8/13) ISP, 58% (7/12) ISP-ISP, 63% (10/16) ISP-transformed, 54% (7/13) SCC-ISP and 5% (1/19) SCC de novo. Protein expression of pEGFR was detected in 56% (5/9) ISP, 75% (6/8) ISP-ISP, 47% (7/15) ISP-transformed, 42% (5/12) SCC-ISP and 71% (10/14) SCC de novo. We observed an inverse correlation between EGFR exon 20 mutation and pEGFR expression (p=0.034). Overall survival was significantly better for SCC-ISP compared with SCC de novo (2-year survival 54% and 29% respectively; p=0.030). Conclusions EGFR exon 20 mutations occurred in a high frequency, both for cases with single or multiple ISP and for ISP-transformed, making it a characterizing genetic abnormality for ISP in general. This result also means that the presence of a EGFR exon 20 mutations is not of value as a risk marker for malignant progression. Among the SCC, EGFR exon 20 mutations were notably less and pEGFR expression more frequent in SCC de novo compared to SCC-ISP, suggesting that the EGFR signaling pathway is important in both types of SCC, albeit activated in a different manner. Patients with SCC-ISP had a more favorable clinical course, however, neither EGFR exon 20 mutations nor pEGFR expression demonstrated prognostic value. Nevertheless, EGFR exon 20 mutations can be targeted with specific inhibitors and may be of value for adjuvant therapy for SCC as well as ISP that are difficult to manage. Citation Format: Virginia N. Cabal, Marta Menéndez, Sira Potes-Ares, Blanca Vivanco, Laura Suárez-Fernández, Cristina Riobello, Rocío García-Marín, Fernando López, José Luís Llorente, Mario Hermsen. EGFR mutation and protein expression analysis in sinonasal inverted papilloma and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3325.

Abstract 309: FAK blockade enhances antitumor effect of BTK inhibitor in esophageal squamous cell carcinoma via EGFR-ERK-Akt pathway inhibition

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is a fatal disease with poor prognosis and lack of effective targeted therapy. Recently, some researches have shown that BTK can be a novel therapeutic target for esophageal cancer, while the mechanism remains unknown. In addition, FAK is one of the important factors to promote the metastasis of esophageal cancer. In this study, we sought to explore the synergistic antitumor effect and underline mechanism of combining BTK inhibitor Ibrutinib with a novel FAK inhibitor APG-2449 for esophageal squamous cell carcinoma. Methods: Cell viability and growth inhibition were determined by CCK-8 assay and colony formation assay. Migration ability was detected by Transwell assay. EMT markers and ki67 were measured by RT-qPCR. Cell cycle analysis and apoptosis were confirmed by flow cytometry. Western Blot analysis and siRNA interference for mechanism exploration. ESCC xenograft models were established to evaluated the synergistic antitumor effect in vivo. Results: We found that ESCC cell lines harbor EGFR or c-myc-amplified exhibited more sensitive to Ibrutinib inhibition. Moreover, Ibrutinib combined with APG-2449 showed synergistic inhibition of cell proliferation in ESCC cell lines. By analyzing data from the Gene Expression Profiling Interactive Analysis database, we found that EGFR expression positively correlated with FAK expression in ESCC samples. The combination of Ibrutinib and APG-2449 exerted obviously synergistic apoptosis induction and colony formation inhibition. Furthermore, we found that Ibrutinib combined with APG-2449 could induce more cancer cells arrested in G1/S phase. In addition, the co-treatment of Ibrutinib and APG-2449 could significantly suppress ESCC cells migration by upregulating E-cadherin. Ibrutinib alone could down-regulate the protein expression of p-EGFR and p-ERK. Moreover, combination treatment significantly reduced the expression of p-Akt and p-c-myc, and upregulated P21 and PUMA expression. In ESCC xenotransplantation models, single therapy with either Ibrutinib or APG-2449 was equivalent in delaying tumor growth, while the combination therapy demonstrated more significantly tumor growth suppression. Conclusions: Taken together, our data strongly suggest that the combination of FAK inhibitor APG-2449 and Ibrutinib provide an effective and precise therapeutic strategy for treating esophageal squamous cell carcinoma. Citation Format: Qiu-Yun Luo, Su-Na Zhou, Wen-Tao Pan, Meng-xian Pan, Lu-ping Yuan, Lin Zhang, Xiang-Lei Yan, Yu-xin Zhang, Jian Sun, Miaozhen Qiu, Da-Jun Yang. FAK blockade enhances antitumor effect of BTK inhibitor in esophageal squamous cell carcinoma via EGFR-ERK-Akt pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 309.

Abstract 539: MAEL promotes colorectal cancer cell growth and migration by activating EGFR pathway

Abstract Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system. It is generally accepted that CRC pathogenesis is a long-term process involving accumulation of multiple genetic alterations. Herein, we aim to explore the molecular biological mechanisms of CRC initiation and progression. We have been committed to investigating MAEL function in digestive neoplasms and have found MAEL is a new candidate oncogene. Previously, overexpression of MAEL was frequently detected in CRC tissues and significantly associated with poor 5-year outcome. By MTT assay and transwell assay, functional study demonstrated that overexpression of MAEL promoted cell growth and cell migration in DLD1 and THC8307 cell lines. Furthermore overexpression of MAEL in DLD1 cell line could promote tumor formation in nude mice. Further study found that MAEL overexpression in DLD1 and THC8307 elevated the protein expression of p-EGFR, p-Raf-1, p-ERK 1/2, p-PI3K and p-AKT. These data indicated that MAEL enhanced the activity of EGFR/Ras/Raf/MEK/ERK1/2 and EGFR/PI3K/Akt pathways. However, more effort is needed to investigate MAEL function and how MAEL activate EGFR pathways. Citation Format: Lulu Liu, Zhou Tong, Weiqin Jiang, Yi Zheng, Peng Zhao, Weijia Fang. MAEL promotes colorectal cancer cell growth and migration by activating EGFR pathway [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 539. doi:10.1158/1538-7445.AM2017-539

Phosphorylated Epidermal Growth Factor Receptor Expression Is Associated With Clinicopathologic Parameters and Patient Survival in Mobile Tongue Squamous Cell Carcinoma

Phosphorylated epidermal growth factor receptor (pEGFR) activates several signaling pathways, resulting in tumor-promoting cellular activities, and has been implicated in malignant transformation and disease progression. The present study evaluated the clinical significance of pEGFR protein expression in mobile tongue squamous cell carcinoma (SCC). The present cohort study included 48 patients with mobile tongue SCC. We evaluated whether pEGFR immunohistochemical protein expression is associated with clinical variables and patient outcome. Of the 48 patients included in the present cohort study, 25 were men and 23 were women. The median patient age was 60years (interquartile range 53 to 72). pEGFR protein expression was significantly increased in well-differentiated tumors compared with poorly differentiated tumors (P=.001). Elevated pEGFR protein expression was significantly more frequently observed in mobile tongue SCC cases with a well-defined tumor shape and an earlier disease stage (P= .010 and P=.019, respectively). Patients with mobile tongue SCC presenting with elevated pEGFR expression had longer overall and disease-free survival times compared with those with low pEGFR expression (P= .015 and P= .006, respectively; log-rank test). On multivariate analysis, pEGFR expression proved to be an independent prognostic factor of both overall and disease-free survival (P= .008 and P= .044, respectively; Cox regression analysis). The results of the present study support evidence that the pEGFR signaling pathway might be implicated in the malignant transformation of mobile tongue SCC. Additional studies are recommended to validate whether pEGFR could be used as a potential biomarker and therapeutic target in mobile tongue SCC.

Low expression of the E3 ubiquitin ligase CBL confers chemoresistance in human pancreatic cancer and is targeted by epidermal growth factor receptor inhibition.

Expression of CBL, an ubiquitin ligase, is decreased in 60% of human pancreatic ductal adenocarcinomas (PDAC) and is associated with shorter overall survival. We sought to determine how low CBL directly contributes to clinically more aggressive PDAC. Human PDACs were stained for CBL, pEGFR, and EGFR. CBL-low was modeled in PDAC cells (Panc-1, L3.6pl, and AsPC-1) via transient transfection (siRNA) or stable knockdown (shRNA). Cell viability and apoptosis were measured by MTT assays and FACS. Immunoblot and a phospho-receptor tyrosine kinase (pRTK) array were used to probe signal transduction. NOD-scid-IL2Rγ(null) mice were subcutaneously implanted with PDAC or PDAC(CBL-low) cells on opposite flanks and treated with gemcitabine ± erlotinib for ≥4 weeks. There was an inverse correlation between CBL and pEGFR protein expression in 12 of 15 tumors. CBL knockdown increased PDAC resistance to gemcitabine and 5-fluorouracil (5-FU) by upregulating pEGFR (Y1068), pERK, and pAKT. A pRTK array of PDAC(CBL-low) cells revealed additional activated tyrosine kinases but all to a much lower magnitude than EGFR. Increased chemoresistance from low CBL was abrogated by the EGFR inhibitor erlotinib both in vitro and in vivo. Erlotinib+gemcitabine-treated PDAC(CBL-low) cells exhibited greater apoptosis by cleaved PARP, caspase-3, and Annexin V/PI. Low CBL causes chemoresistance in PDAC via stress-induced EGFR activation that can be effectively abrogated by EGFR inhibition. These results suggest that dysregulation of ubiquitination is a key mechanism of EGFR hyperactivation in PDAC and that low CBL may define PDAC tumors likely to respond to erlotinib treatment.

Nimbolide inhibits invasion and migration, and down-regulates uPAR chemokine gene expression, in two breast cancer cell lines.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women, worldwide. Urokinase type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis of breast cancer. Nimbolide is a potent cytotoxic limnoid isolated from Azadirachta indica. Our previous studies have shown that nimbolide elicits pleiotropic effects on breast cancer cells; however, its roles in invasion and migration have not previously been fully elucidated. Protein expression of pEGFR, VEGFR, NFκB, IKKα, IKKβ, MMP-2, MMP-9 and TIMP-2 were analysed by western blotting. We also analysed expressions of uPA, uPAR genes and chemokines by real-time PCR. Breast cancer cell invasion was assessed by transwell invasion assay and cell migration analysed by scratch wound healing assay. Our results showed that reduced protein expression of pEGFR, VEGFR, NFκB, IKKα, β, MMP-2, MMP-9 and TIMP-2 was higher in nimbolide-treated breast cancer cells. mRNA expression of uPA, uPAR, chemokines and their receptors were also significantly reduced in response to nimbolide treatment. Nimbolide inhibited breast cancer cell migration and invasion as shown in transwell invasion and wound healing assays. These results clearly proved inhibitory effects of nimbolide on tumour cell invasion and migration by down-regulating proteins critically involved in regulation of cell invasion and metastasis, suggesting a possible therapeutic role of nimbolide for breast cancer.

Effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells

The study investigated the effects of pemetrexed, gefitinib, and their combination on human colorectal cancer cells. Six human colorectal cancer cells were exposed to pemetrexed, gefitinib, and their combination. Antitumor effects were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Thymidylate synthase (TS) mRNA expression and EGFR mutation were studied by real-time RT-PCR and DNA sequence determination. Pharmacological interaction was studied using the combination index method. Cell cycle distribution and apoptosis were determined by flow cytometry. Activity assay was performed to assess the effects of drugs on TS activity, and Western blot was performed to assess the protein expression of pEGFR, pAKT, and pERK 1/2. Six colorectal cancer cells are all sensitive to pemetrexed, and TS gene expression of cells was negatively related to pemetrexed sensitivity. The cytotoxic synergism was observed in concurrent pemetrexed combined with gefitinib and sequential pemetrexed followed by gefitinib. The combination of pemetrexed and gefitinib modulated cell cycle and induced apoptosis. Pemetrexed combined with gefitinib decreased TS mRNA expression and in situ activity. Pemetrexed induced an EGFR-mediated activation of the phosphatidylinositol 3-kinase/AKT and ERK pathway, which was inhibited by gefitinib. Pemetrexed is a promising agent, and pemetrexed combined with gefitinib has a significantly synergistic effect on colorectal cancer cells, which seems to present a strategy of pemetrexed combined with EGFR-TKIs in colorectal cancer treatment.

Activation of the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway in human cholesteatoma epithelium

Cholesteatoma is a benign keratinizing squamous epithelial lesion characterized by the hyper-proliferation of keratinocytes with abundant production of keratin debris in the middle ear. The epidermal growth factor receptor (EGFR)/Akt/nuclear factor-kappa B (NF-κB)/cyclinD1 signaling pathway is one of the most important pathways in regulating cell survival and proliferation. We hypothesized that the EGFR/Akt/NF-κB/cyclinD1 signaling pathway may be activated and involved in the cellular hyperplasia mechanism in acquired cholesteatoma epithelium. Immunohistochemical staining of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt), activated NF-κB and cyclinD1 protein was performed in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium. Protein expression of p-EGFR, p-Akt, activated NF-κB and cyclinD1 in cholesteatoma epithelium was significantly increased when compared with normal EAC epithelium (p < 0.01). In cholesteatoma epithelium, a significant positive association was observed between p-EGFR and p-Akt expression and between the expressions of p-Akt and NF-κB, NF-κB and cyclinD1, respectively (p < 0.01). No significant relationships were observed between the levels of investigated proteins and the degree of bone destruction (p > 0.05). The increased protein expression of p-EGFR, p-Akt, NF-κB and cyclinD1 and their associations in cholesteatoma epithelium suggest that the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway is active and may be involved in the regulatory mechanisms of cellular hyperplasia in cholesteatoma epithelium.

Epigallocatechin gallate sensitizes CAL-27 human oral squamous cell carcinoma cells to the anti-metastatic effects of gefitinib (Iressa) via synergistic suppression of epidermal growth factor receptor and matrix metalloproteinase-2

Human head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related death during the last decade due to its related metastasis and poor treatment outcomes. Gefitinib (Iressa), a tyrosine kinase inhibitor has been reported to reduce the metastatic abilities of oral cancer. Previous studies have shown that epigallocatechin gallate (EGCG), a green tea polyphenol, possesses cancer chemopreventive and anticancer activity. However, the mechanisms involved in the suppression of invasion and metastasis of human oral cancer cells following co-incubation with gefitinib and EGCG remain poorly understood. In the present study, we attempted to investigate the synergistic effects of a combined treatment of gefitinib and EGCG in CAL-27 cells in vitro and to elucidate the underlying molecular mechanisms associated with the supression of cell migration and invasion. In the present study, we found that the individual treatments or the combined treatment of gefitinib and EGCG synergistically inhibited the invasion and migration of CAL-27 cells using Transwell invasion and wound-healing scratch assays, respectively. Similarly, gefitinib in combination with EGCG synergistically attenuated enzymatic activity and the protein expression of MMP-2 in CAL-27 cells. Furthermore, individual or combined treatment with EGCG and gefitinib suppressed the protein expression of p-EGFR and the phosphorylated protein levels of ERK, JNK, p38 and AKT and displayed inhibitory effects on metastatic ability of CAL-27 cells. Combined effects of EGCG and gefitinib-altered anti-metastatic actions for related gene expression were observed using DNA microarray analysis. Importantly, EGCG sensitizes CAL-27 cells to gefitinib-suppressed phosphorylation of epidermal growth factor receptor (EGFR in vitro. Taken together, our results suggest that the synergistic suppression of the metastatic ability of CAL-27 cells after EGCG and gefitinib individual or combined treatment are mediated through mitogen-activated protein kinase (MAPK) signaling. Our novel findings provide potential insights into the mechanism involved with synergistic responses of gefitinib and EGCG against the progression of oral cancer.

Abstract 3523: Biochanin A enhances anticancer activity of temozolomide in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most common and most lethal form of primary brain tumor, classified as a Grade IV astrocytoma by World Health Organization (WHO). The poor prognosis of patients is a result of an extremely invasive and aggressive nature of this tumor. Temozolomide, an anticancer agent currently approved for the treatment of glioblastoma is only modestly effective as a single agent in enhancing patient survival. Therefore, it is imperative to design therapies which are safe and can more effectively target the progression of glioblastoma. Our laboratory has been investigating the anticancer properties of biochanin A, a bioactive isoflavone found in red clover and soy products. This compound shows anticancer potential against breast, oral, pancreatic and brain cancers. Previous studies from our laboratory show that biochanin A inhibits invasion in GBM cells. In this study we investigated the anticancer potential of combination of biochanin A and temozolomide on U-87 MG cells (human glioblastoma cell line). We hypothesized that this combination will enhance the efficacy of temozolomide towards inhibiting invasion and proliferation of glioblastoma cells. We performed colony formation assay to assess the cytotoxicity of treatment(s). Biochanin A (20μM and 70μM) enhances the effect of temozolomide in decreasing the number of colonies formed as determined by the colony formation assay. Further, we determined the effect of temozolomide (100μM) alone and in combination with biochanin A (20μM and 70μM) on cell signaling, invasive processes and transcription factors. Western blot analysis shows a decrease in the protein expression of p-EGFR, p-ERK, uPAR, MMP-2. Our results implicate that combining biochanin A with temozolomide can improve the anticancer effect of temozolomide on glioblastoma cells. These findings help us better understand how biochanin A can be used to seek new and more effective combination therapy. Further studies are aimed to elucidate the mechanism(s) by which this combination inhibits invasion of U-87 MG cells. This study was supported by in parts from NIH INBRE GRANT P20RR016454. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3523. doi:10.1158/1538-7445.AM2011-3523

Abstract 1742: Evaluating the prognostic value of EGFR by p16/HPV status in patients with squamous cell carcinoma of the head and neck treated with concurrent chemoradiation

Abstract Background: Overexpression of the epidermal growth factor receptor (EGFR) has been correlated with a worse prognosis in patients with squamous cell carcinoma of the head and neck (SCCHN), however the prognostic value of EGFR has not been fully evaluated in the context of human papillomavirus (HPV) status. Methods: Records from 91 consecutive SCCHN patients treated with concurrent chemoradiation at the Tom Baker Cancer Centre between August 2000 and March 2005 were reviewed. Triplicate 0.6 mm cores from available pre-treatment formalin-fixed paraffin-embedded (FFPE) biopsies were assembled into a tissue microarray. Immunofluorescence combined with automated quantitative analysis (AQUA™) was used to evaluate EGFR and p16 protein expression. Phosphorylated EGFR (pEGFR) was assessed by proximity ligation assay. HPV infection status was determined by colorimetric in-situ hybridization using the INFORM HPV Family 16 Probe (CISH). PCR was used to detect HPV DNA. EGFR and pEGFR were correlated with clinical outcomes according to p16-status and HPV-status. Results: FFPE biopsies were available from 49 patients (54%). Patient characteristics were as follows: mean age 55 years, 82% male, 49% oropharyngeal primaries. The complete response rate was 88%. With a median follow-up of 42 months (range 3 to 60), the 5 year overall survival (OS) was 54%. EGFR and pEGFR protein expression were classified as high in 14 (29%) and 13 (27%) tumours respectively. 27 tumours were p16 positive; 30 were HPV positive by either PCR or CISH. p16 was strongly correlated with HPV status (Spearman r=0.80). In univariate analyses, tumours with high EGFR, low p16, and HPV negative were associated with significant 3-, 7-, and 5-fold increases, respectively, in locoregional recurrence, recurrence at any site, disease-specific mortality and worse OS. pEGFR did not predict outcome. Neither EGFR nor pEGFR predicted response. After adjusting for age, smoking status, T- and N-stage, p16, and HPV there was no relationship between EGFR and outcome. p16 and EGFR were inversely related (Spearman r=-0.3). Conclusions: In our small cohort of patients, EGFR did not have prognostic value after correction for p16 / HPV status. 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 1742.

Abstract 3505: EGFR-targeted hybrid plasmonic magnetic nanoparticles induce autophagy and apoptosis through DNA damage in non-small cell lung cancer cells

Abstract Nanoparticles represent a novel class of agents for cancer treatment. In the present study, we examined the effects of epidermal growth factor receptor (EGFR)-targeted hybrid plasmonic magnetic nanoparticles (NP) on non-small cell lung cancer (NSCLC) cells. The nanoparticles (NP) consisted of a paramagnetic iron core that is surrounded by a gold layer and is functionalized with monoclonal anti-EGFR antibody (C225) or control rabbit IgG antibody (IgG). The nanoparticles were tested against a panel of human lung cancer cells that were mutant (HCC827, H1819), wild-type (H1299) or null (H520) for EGFR and compared to EGFR-positive normal fibroblasts (MRC9, WI38) and normal human bronchial epithelial (NHBE) cells. Treatment of tumor cells with C225-NP resulted in a strong and significant inhibitory effect of cell growth compared to normal cells. C225-NP-mediated inhibitory effect was observed only in EGFR-positive tumor cells (HCC827, H1819, H1299) but not in EGFR-null H520 cells. EGFR-mutant cells (HCC827) were the most sensitive to C225-NP. No significant growth inhibitory effects were observed in IgG-NP-treated cells when compared to untreated cells. Molecular analysis showed C225-NP selectively inhibited phosphorylated p-EGFR protein expression and EGFR-mediated signal transduction resulting in induction of autophagy followed by apoptosis. Autophagy occurred as early as twenty-four hours after treatment with an increase in LC3-II content and autophagic vacuoles observed by Western blotting and electron microscopy respectively. Apoptosis as determined by flow cytometry and Western blotting showed an increase in the number of sub-G1 population cells and cleavage of caspase-3 and PARP in C225-NP-treated cells. Optical images determined by dark-field microscopy showed an increase in the concentration of C225-NP bound to EGFR-expressing NSCLC cells but not EGFR-null NSCLC cells. C225-NP exhibited 2-7 fold higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody. Free C225 antibody did not induce autophagy in cells. Investigation into how C225-NP but not C225 antibody or NP-alone triggers autophagy and initiates apoptosis showed induction of a DNA damage response as evidenced by the formation of γH2AX foci in C225-NP-treated cells. Minimal to no γH2AX foci were observed in cells that were untreated, treated with C225 antibody or NP-alone or treated with IgG-NP. Our data show that EGFR specific NP selectively induced cytotoxic effects in EGFR-expressing lung cancer cells via a novel cell-death mechanism that is not characteristic of the free antibody thus increasing efficacy of therapy. 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 3505.