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Supplementary Figures 1-5 from Sorafenib Inhibits the Hepatocyte Growth Factor–Mediated Epithelial Mesenchymal Transition in Hepatocellular Carcinoma

Purpose: BIBF 1120 is a potent, orally available triple angiokinase inhibitor that inhibits VEGF receptors (VEGFR) 1, 2, and 3, fibroblast growth factor receptors, and platelet-derived growth factor receptors. This study examined the antitumor effects of BIBF 1120 on hepatocellular carcinoma (HCC) and attempted to identify a pharmacodynamic biomarker for use in early clinical trials.Experimental Design: We evaluated the antitumor and antiangiogenic effects of BIBF 1120 against HCC cell line both in vitro and in vivo. For the pharmacodynamic study, the phosphorylation levels of VEGFR2 in VEGF-stimulated peripheral blood leukocytes (PBL) were evaluated in mice inoculated with HCC cells and treated with BIBF 1120.Results: BIBF 1120 (0.01 μmol/L) clearly inhibited the VEGFR2 signaling in vitro. The direct growth inhibitory effects of BIBF 1120 on four HCC cell lines were relatively mild in vitro (IC50 values: 2–5 μmol/L); however, the oral administration of BIBF 1120 (50 or 100 mg/kg/d) significantly inhibited the tumor growth and angiogenesis in a HepG2 xenograft model. A flow cytometric analysis revealed that BIBF 1120 significantly decreased the phosphotyrosine (pTyr) levels of VEGFR2+CD45dim PBLs and the percentage of VEGFR2+pTyr+ PBLs in vivo; the latter parameter seemed to be a more feasible pharmacodynamic biomarker.Conclusions: We found that BIBF 1120 exhibited potent antitumor and antiangiogenic activity against HCC and identified VEGFR2+pTyr+ PBLs as a feasible and noninvasive pharmacodynamic biomarker in vivo. Clin Cancer Res; 17(6); 1373–81. ©2010 AACR.

Supplementary Figure Legends from Sorafenib Inhibits the Hepatocyte Growth Factor–Mediated Epithelial Mesenchymal Transition in Hepatocellular Carcinoma

Supplementary Figures S1-S3; Supplementary Tables S1-S2.

The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells. Mol Cancer Ther; 10(1); 169–77. ©2011 AACR.

Supplementary Figure Legends 1-2 from mTOR Signal and Hypoxia-Inducible Factor-1α Regulate CD133 Expression in Cancer Cells

Supplementary Figures 1-2 from mTOR Signal and Hypoxia-Inducible Factor-1α Regulate CD133 Expression in Cancer Cells

The underlying mechanism regulating the expression of the cancer stem cell/tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. Here, we found that the inhibition of mTOR signaling up-regulated CD133 expression at both the mRNA and protein levels in a CD133-overexpressing cancer cell line. This effect was canceled by a rapamycin-competitor, tacrolimus, and was not modified by conventional cytotoxic drugs. We hypothesized that hypoxia-inducible factor-1α (HIF-1α), a downstream molecule in the mTOR signaling pathway, might regulate CD133 expression; we therefore investigated the relation between CD133 and HIF-1α. Hypoxic conditions up-regulated HIF-1α expression and inversely down-regulated CD133 expression at both the mRNA and protein levels. Similarly, the HIF-1α activator deferoxamine mesylate dose-dependently down-regulated CD133 expression, consistent with the effects of hypoxic conditions. Finally, the correlations between CD133 and the expressions of HIF-1α and HIF-1β were examined using clinical gastric cancer samples. A strong inverse correlation (r = −0.68) was observed between CD133 and HIF-1α, but not between CD133 and HIF-1β. In conclusion, these results indicate that HIF-1α down-regulates CD133 expression and suggest that mTOR signaling is involved in the expression of CD133 in cancer cells. Our findings provide a novel insight into the regulatory mechanisms of CD133 expression via mTOR signaling and HIF-1α in cancer cells and might lead to insights into the involvement of the mTOR signal and oxygen-sensitive intracellular pathways in the maintenance of stemness in cancer stem cells. [Cancer Res 2009;69(18):7160–4]

The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells. Mol Cancer Ther; 10(1); 169–77. ©2011 AACR.

Purpose: The objective of this study was to evaluate the efficacy and safety of two doses of pemetrexed supplemented with folic acid and vitamin B12 in pretreated Japanese patients with advanced non-small cell lung cancer (NSCLC). Experimental Design: Patients with an Eastern Cooperative Oncology Group performance status 0 to 2, stage III or IV, and who received previously one or two chemotherapy regimens were randomized to receive 500 mg/m2 pemetrexed (P500) or 1,000 mg/m2 pemetrexed (P1000) on day 1 every 3 weeks. The primary endpoint was response rate. Results: Of the 216 patients evaluable for efficacy (108 in each arm), response rates were 18.5% (90% confidence interval, 12.6-25.8%) and 14.8% (90% confidence interval, 9.5-21.6%), median survival times were 16.0 and 12.6 months, 1-year survival rates were 59.2% and 53.7%, and median progression-free survival were 3.0 and 2.5 months for the P500 and P1000, respectively. Cox multiple regression analysis indicated that pemetrexed dose was not a significant prognostic factor. Drug-related toxicity was generally tolerable for both doses; however, the safety profile of P500 showed generally milder toxicity. Main adverse drug reactions of severity grade 3 or 4 were neutrophil count decreased (20.2%) and alanine aminotransferase (glutamine pyruvic transaminase) increased (15.8%) in P500 and neutrophil count decreased (24.3%), WBC count decreased (20.7%), and lymphocyte count decreased (18.0%) in P1000. One drug-related death from interstitial lung disease occurred in the P500. Conclusion: P500 and P1000 are similarly active with promising efficacy and acceptable safety outcomes in pretreated patients with NSCLC. These results support the use of P500 as a second- and third-line treatment of NSCLC.

Purpose: Irinotecan and cisplatin (IP) significantly improved survival compared with etoposide and cisplatin (EP), in patients with extensive-stage small cell lung cancer (SCLC) in a previous Japan Clinical Oncology Group (JCOG) randomized trial. JCOG9903 was conducted to evaluate the safety of sequentially given IP following concurrent EP plus twice-daily thoracic irradiation (TRT) for the treatment of limited-stage SCLC (LSCLC). Experimental Design: Between October 1999 and July 2000, 31 patients were accrued from 10 institutions. Thirty patients were assessable for toxicity, response, and survival. Treatment consisted of etoposide 100 mg/m2 on days 1 to 3, cisplatin 80 mg/m2 on day 1, and concurrent twice-daily TRT of 45 Gy beginning on day 2. The IP regimen started on day 29 and consisted of irinotecan 60 mg/m2 on days 1, 8, and 15 and cisplatin 60 mg/m2 on day 1, with three 28-day cycles. Results: There were no treatment-related deaths. The response rate was 97% (complete response, 37%; partial response, 60%). Median overall survival was 20.2 months; 1-, 2-, and 3-year survival rates were 76%, 41%, and 38%, respectively. Of the 24 patients who started the IP regimen, 22 received two or more cycles. Hematologic toxicities of grade 3 or 4 included neutropenia (67%), anemia (50%), and thrombocytopenia (4%). Nonhematologic toxicities of grade 3 or 4 included diarrhea (8%), vomiting (8%), and febrile neutropenia (8%). Of the 20 patients with recurrence, none had local recurrence alone and only two had both local and distant metastasis as the initial sites of disease progression. Conclusions: IP following concurrent EP plus twice-daily TRT is safe with acceptable toxicities. A randomized phase III trial comparing EP with IP following EP plus concurrent TRT for LSCLC is ongoing (JCOG0202).

Purpose: We investigated single-nucleotide polymorphisms of the cytidine deaminase gene (CDA), which encodes an enzyme that metabolizes gemcitabine, to clarify the relationship between the single-nucleotide polymorphism 208G>A and the pharmacokinetics and toxicity of gemcitabine in cancer patients treated with gemcitabine plus cisplatin. Experimental Design: Six Japanese cancer patients treated with gemcitabine plus cisplatin were examined. Plasma gemcitabine and its metabolite 2′,2′-difluorodeoxyuridine were measured using an high-performance liquid chromatography method, and the CDA genotypes were determined with DNA sequencing. Results: One patient, a 45-year-old man with pancreatic carcinoma, showed severe hematologic and nonhematologic toxicities during the first course of chemotherapy with gemcitabine and cisplatin. The area under the concentration-time curve value of gemcitabine in this patient (54.54 μg hour/mL) was five times higher than the average value for five other patients (10.88 μg hour/mL) treated with gemcitabine plus cisplatin. The area under the concentration-time curve of 2′,2′-difluorodeoxyuridine in this patient (41.58 μg hour/mL) was less than the half of the average value of the five patients (106.13 μg hour/mL). This patient was found to be homozygous for 208A (Thr70) in the CDA gene, whereas the other patients were homozygous for 208G (Ala70). Conclusion: Homozygous 208G>A alteration in CDA might have caused the severe drug toxicity experienced by a Japanese cancer patient treated with gemcitabine plus cisplatin.

Non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show a dramatic response to EGFR tyrosine kinase inhibitors (EGFR TKIs). However, most patients have a relapse within 1 year after the initiation of therapy. An EGFR secondary mutation T790M has been observed in approximately 50 % of such lung cancers that acquire resistance to EGFR TKIs. A critical question is whether the T790M mutation occurs as a result of treatment with EGFR TKIs or whether it exists prior to treatment and is selected during the course of therapy. However, its assessment can be challenging due to limited tissue availability and low sensitivity of the previously available methods. Here, we sought to detect the T790M using colony hybridization (CH) assay. This assay is able to detect mutant allele of 0.01 % in the background of wild-type allele. We assessed the T790M mutation in pretreatment specimens from 39 EGFR-TKI-treated patients with activating EGFR mutations. The T790M mutation was detected by CH in 31 of 39 patients (79 %). Median time to treatment failure (TTF) was 9 months in patients with pretreatment T790M, whereas it was 7 months in patients without the T790M mutation (P = 0.44). When we subdivided the patients with T790M into a strong and a modest positive subgroups in terms of the frequency of positive signals observed by our colony hybridization technique, seven patients with strong positivity had a TTF significantly longer than 8 patients without T790M (P = 0.0097) and 24 patients with modest positivity (P = 0.0019). Thus, the use of our highly sensitive method showed that a subgroup of patients with the EGFR mutation harbor rare T790M alleles before exposure to EGFR TKIs. However, contrary to our expectation, such patients had longer TTF. 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 1209. doi:1538-7445.AM2012-1209

Cancer cells that expressed mutant EGFR are more sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs) than that expressed wild type EGFR. To elucidate difference of the characteristics between wild type and 15 bp deletion mutant EGFR, we explored the difference of EGFR-binding proteins using respective stable transfectants, 293_pEGFR and 293_p≤15. As the result, we detected heat shock protein 70 (HSP70) specifically binds to the mutant EGFR but wild type EGFR. To examine whether HSP70 influenced EGFR-TKI sensitivity, we examined the effect of HSP70 inhibition. [Methods] HSP70 siRNA was exposed to the cells for 2 days. Cytotoxicity of the drugs was measured by MTT assay. The drug binding to EGFR was measured by [14C]gefitinib. [Results] Suppression of HSP70 by siRNA increased gefitinib and erlotinib sensitivities and enhanced gefitinib binding to this receptor, in 293_p≤15 cells. Same phenomena were observed when HSP70 was inhibited by HSP70 specific inhibitor, 2-phenylethynesulfonamide (PES). PES inhibits HSP70 binding to the mutant EGFR. Interestingly, this compound enhanced the EGFR autophosphorylation and c-Cbl binding to the receptor in 293_p≤15 cells. These results suggest that HSP70 may modulate EGFR activity and may cause EGFR-TKIs-resistance due to inhibit drug binding to the mutant EGFR. HSP70 inhibitor may possibly enhance the efficacy of EGFR-TKIs. 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 1900. doi:1538-7445.AM2012-1900

Epithelial-Mesenchymal Transition (EMT) has been classified as a unique process by which epithelial cells undergo mesenchymal phenotype leading to increased motility and invasion. The aim of this study was to elucidate the biological functions of Slug/Snail2 in lung cancer cells. We introduced Slug gene into several lung cancer cell line (A549, Ma1, and H1299) and established stable cell lines. Overexpression of Slug clearly mediated the EMT-related morphological changes including the cell scattering and the elongation of cell-shape. Real-time RT PCR and Western blot demonstrated that overexpression of Slug markedly downregulated the mRNA levels and protein levels of E-cadherin, while mesenchymal marker, N-cadherin, fibronectin 1 and vimentin, were upregulated. In addition to these changes, Slug enhanced the cellular migration activity and the cellular anchorage independent growth activity. Finally, we examined the Slug-mediated EMT on drug sensitivity to several anti-cancer dugs. Interestingly, Slug-overexpressing cells increased the drug sensitivity to only tubulin-binding agents such as vinorelbine, vincristine, and paclitaxel in vitro. Microarray analysis revealed that Slug downregulates tubulin beta 3 or 4 expression. Luciferase promoter assay showed that Slug directly down-regulates tubulin beta 4 expression at the transcription level. These results suggested that Slug-mediated sensitivity to tubulin-binding agents may be involved in expression changes of tubulin beta 3 or 4.In conclusion, we found that overexpression of Slug mediates EMT and change of drug sensitivity to tubulin-binding agents, presumably via downregulations of tubulin beta 3 and 4 in lung cancer cells. 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 3424. doi:10.1158/1538-7445.AM2011-3424

[Purpose] Non small lung cancer (NSCLC) cells that expressed mutant EGFR are more sensitive to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) than that expressed wild type EGFR. Recent clinical trials revealed that more than 70% NSCLC patients that expressed mutant EGFR showed sensitivity to EGFR-TKIs, such as gefitinib and erlotinib. To elucidate the mechanism of this hypersensitivity, we explored the difference of EGFR-binding proteins between wild type EGFR and 15 bp deletion mutant EGFR using respective stable transfectants. [Methods] Wild type EGFR and a 15 bp deletion mutant EGFR plasmids were transfected into HEK293 cells. The stable transfectant cells were established, and were designated 293_pEGFR and 293_pΔ15, respectively. Cells were incubated in a medium with/without 10 ng/ml of TGFα for 1 h and lysed. Cell lysate was precleared by centrifugation at 15,000 rpm for 15 min. The supernatant was mixed with a polyclonal EGFR antibody for 1 h and EGFR was immunoprecipitated by Protein A-Sepharose. After adequate washing, coprecipitated proteins that bound to EGFR were eluted and separated by 2D-PAGE (Immobiline DryStrip (pH3-10 NL, 7 cm) and 10% SDS-PAGE). Proteins were visualized by silver staining and identified by LC-MS/MS. HSP70 siRNA was transfected into the cells by lipofection method. Sensitivity to gefitinib was measured by MTT assay. EGFR binding affinity to gefitinib was measured using [14C] gefitinib. [Results and Discussion] We detected several EGFR binding proteins. Among these proteins, one candidate (lesser binding to wild type EGFR than the mutant EGFR) was identified as HSP 70 by LC-MS/MS. The total amount of HSP70 protein was not different between 293_pEGFR and293_pΔ15 cells. A specific binding of this protein to the mutant EGFR was confirmed by Western blotting analysis. This binding was not modulated by EGFR activation. Knockdown of HSP 70 protein by a specific siRNA enhanced gefitinib sensitivity in the mutant EGFR transfectant. These results suggest that HSP 70 may decrease sensitivity to EGFR-TKIs in the cells that expressed 15 bp deletion mutant EGFR. There is a possibility that HSP70 overexpression might be a newly resistant mechanism to EGFR-TKIs in NSCLC patients that expressed mutant EGFR. 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 699. doi:10.1158/1538-7445.AM2011-699

Epithelial-Mesenchymal Transition (EMT) has been classified as a unique process by which epithelial cells undergo remarkable morphologic changes characterized by a transition from epithelial cobblestone phenotype to elongated fibroblastic phenotype (mesenchymal phenotype) leading to increased motility and invasion. Accumulating evidence indicates that EMT-inducible transcription factors such as Snail homologues (Snail1, 2, and 3) and several basic helix-loop-helix factors Twist, are poor prognostic factors and associated with chemoresistance in oncology. The aim of this study was to investigate the involvement of SNAI-mediated EMT and its malignant potential in lung cancer cells. We introduced SNAI1 or SNAI2 gene into A549 or PC-9 lung cancer cell lines and established stable cell lines A549/GFP, /SNAI1, /SNAI2, PC-9/GFP, /SNAI1 and /SNAI2. Overexpression of SNAI1 and SNAI2 clearly mediated the EMT-related morphological changes including the cell scattering and the elongation of cell-shape. Real-time RT PCR demonstrated that overexpression of SNAI1 and SNAI2 markedly down-regulated the mRNA levels of E-cadherin about 1/2 to 1/100 compared with GFP-expressing control cell lines. The mRNA upregulation of N-cadherin, fibronectin 1 and Vimentin, which changes are characteristic of EMT, were also observed. We are now investigating its oncogenic properties, gene expression changes by microarray analysis and chemoresistance using these stable SNAI1 or SNAI2 transfectants. 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 3313.

Background c-MET, a receptor of hepatocyte growth factor (HGF), is known to be overexpressed in a variety of human cancers. Recently several c-MET inhibitors are being developed as cancer chemotherapy against this compelling molecular target. ARQ 197 is a new small molecule c-MET inhibitor. Since ARQ 197 shows efficient anti-cancer activity without many severe adverse events in Phase I clinical trials, it is expected to be a useful anticancer agent for clinical treatment. This drug is known to bind to c-MET at neighbor area of its ATP binding site and it does not work as an ATP mimic. In this study, we investigated the molecular mechanism of inhibitory effect on c-MET by ARQ 197 in a non-small cell lung cancer cell (NSCLC) line. Materials and Method We established a c-MET-overexpressed NSCLC line, PC-9/MET, by long term exposure of PC-9 cells to EGFR-tyrosine kinase inhibitor. The cytotoxicity of ARQ 197 was measured by MTT assay. The expression of c-MET protein and mRNA were detected by Western blotting analysis and real time RT-PCR method, respectively. In a c-MET degradation analysis, bortezomib was used for inhibition of proteasome. Results ARQ 197 is equally cytotoxic to parental PC-9 and EGFRTKI-resistant PC-9/MET cell line with GI50 values around 200 nM (?). ARQ 197 showed an additive growth- inhibitory effect with erlotinib in PC-9/MET cells as assessed by an Isobologram analysis. In ARQ 197-treated PC-9/MET cells, a clear decrease of phospho c-MET protein as well as phospho AKT and ERK proteins was observed, however, ARQ 197 also induced a down-regulation of c-MET protein in the cells without any effect on c-MET mRNA level. This ARQ 197 induced degradation was reversed by a concomitant treatment with a proteasome inhibitor, bortezomib in the cells. However, bortezomib did not affect cytotoxicity of ARQ 197 in the same cells. Conclusion These data suggest that ARQ 197 may inhibit c-MET through downregulation of this protein in addition to inhibition of its kinase activity. Furthermore, it may enhance c-MET degradation by the ubiquitin/proteasome pathway. These c-MET inhibitory activities by ARQ 197 should be important for the additive combined effect with erlotinib in PC-9/MET cells. 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 1639.

Sorafenib is a multikinase inhibitor with activity against Raf kinase and several receptor tyrosine kinases, including vascular endothelial growth factor receptor2, platelet-derived growth factor receptor. Epithelial mesenchymal transition (EMT) describes a process wherein static epithelial cells lose cell-cell contacts, acquire mesenchymal features and manifest a migratory phenotype, mediating malignant phenotypes for cancer cells. It remains unclear for involvement of Sorafenib against EMT, especially in hepatocellular carcinoma (HCC). Hepatocyte growth factor (HGF) activated HGF/MET signaling pathway in HepG2 and Huh7 cells and resulted in cell scattering and spindle-shaped morphology, changes that are characteristic of EMT. SNAI1 is a key transcription factor known to be involved in EMT. HGF up-regulated SNAI1 and N-cadherin expression, and down-regulated E-cadherin in a dose- and time-dependent manner. Knockdown of SNAI1 by siRNA almost completely inhibited these morphological changes, indicating that SNAI1 was required for HGF-induced EMT in HCC cell lines. Similarly, Sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced SNAI1 expression and these morphological changes at 10 μM of concentration, whereas PI3 kinase inhibitor Wortmannin did not. In conclusion, Sorafenib exerts a potent inhibitory activity against EMT via ERK/SNAI1 in HCC. This activity may provide an additional clinical benefit in patients with HCC. 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 2296.

Background: The mechanisms of resistance to HER2-targeted agents and the means to overcome them have not been fully understood. We investigated the influence of PIK3CA mutations on sensitivity to HER2-targeted agents in naturally derived breast cancer cells. We also tested potential usefulness of various inhibitors against molecules on PI3K signaling pathway. Materials and Methods: We examined the effects of CL-387,785, HER2 tyrosine kinase inhibitor, and trastuzumab, humanized anti-HER2 monocloncal antibody, on cell growth and HER2 signaling in eight breast cancer cell lines showing HER2 amplification and trastuzumab-conditioned BT474 (BT474-TR). We also examined different PI3K pathway inhibitors that were PI3K inhibitor, m-TOR inhibitor, and PI3K/m-TOR dual inhibitor, on the same panel of cell lines. Results: Four cell lines with PIK3CA mutations (E545K and H1047R) were more resistant to trastuzumab than the remaining four without mutations (mean percentage of control with 10 mg/ml trastuzumab: 58% versus 92%; p = 0.010). While PIK3CA-mutant cells were more resistant to CL-387,785 than PIK3CA-wild-type cells (mean percentage of control with 1 M CL-387,785: 21% versus 77%; P = 0.001), CL-387,785 retained activity against BT474-TR. Growth inhibition by trastuzumab and CL-387,785 was more closely correlated with changes in phosphorylation of S6K (correlation coefficient, 0.811) than those of HER2, Akt, or ERK1/2. All HER2-amplified cells showed detectable level of phosphorylated-Akt on serum-starved condition, while cell lines without PI3K pathway enhancement or HER2-amplification did not. Growth of all HER2-amplified cells was inhibited by PI3K pathway inhibitors regardless of PIK3CA genotype. PI3K/m-TOR dual inhibitor had much lower IC50 than the other PI3K pathway inhibitors, suggesting that complete blockade of phosphorylation of Akt and S6K may be required for maximal growth inhibition. Conclusions: PIK3CA mutations are associated with resistance to HER2-targeted agents. S6K phosphorylation is a possibly useful pharmacodynamic marker in HER2-targeted therapy. PI3K inhibitors, especially PI3K/m-TOR dual inhibitor, are potentially effective in overcoming trastuzumab resistance caused by PIK3CA mutations. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A182.