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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 Legend from FOXQ1 Is Overexpressed in Colorectal Cancer and Enhances Tumorigenicity and Tumor Growth

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

Supplementary Figure 1 from FOXQ1 Is Overexpressed in Colorectal Cancer and Enhances Tumorigenicity and Tumor Growth

Supplementary Tables 1-2 from FOXQ1 Is Overexpressed in Colorectal Cancer and Enhances Tumorigenicity and Tumor Growth

Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21CIP1/WAF1 expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 ± 301 versus 1735 ± 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family. Cancer Res; 70(5); 2053–63

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]

After review of the literature, there appears to be no report on the histology of the mandibular nerve fiber distribution. Therefore, using a Wistar rat model, immunohistochemical staining with protein gene product (PGP) and calcitonin gene-related peptide (CGRP) antibody for all nerves and only the pain-sensitive nerves, respectively, was performed. We also statistically compared the nerve distribution density by mandibular region. The section of the mandible from the alveolar crest to the mandibular canal was compartmentalized to several regions. Subsequently, nerve distribution density by region was measured microscopically in both the PGP- and CGRP-positive nerves. Furthermore, the ratio of CGRP- to PGP-positive nerves was measured in each region and statistically compared. In both the PGP- and CGRP-positive nerves, the nerve distribution density significantly increased vertically toward the mandibular canal from the alveolar crest and horizontally toward the periodontal ligament from the periosteum. From the CGRP- to PGP-positive nerve ratio, the pain-sensitive nerve accounted for approximately >70% in each region. Pain would therefore be more likely to develop when surgical invasiveness deepens toward the mandibular canal or periodontal ligament. Therefore, sufficient local anesthetic infiltration and/or combined use of conduction anesthesia or periodontal ligament injection may be required. These results may aid in the development of more effective surgical and anesthetic techniques for mandibular surgery.

To assess the effect of epinephrine-containing local anesthetics on vasoconstriction, we immunohistochemically measured the intravascular lumen area in different regions of the mandible. Twelve male Wistar rats were used. General anesthesia was induced and maintained with sevoflurane. Infiltration anesthesia was performed with 0.2 mL of epinephrine-free 2% lidocaine (E−) near the left mandibular first molar and with 0.2 mL of epinephrine-containing 2% lidocaine (E+) near the right mandibular first molar. After decalcification, the specimens were paraffinized, and thin sections were prepared and immunohistologically stained with an antismooth muscle actin antibody. The intravascular lumen area was measured in the mucosa, periodontal membrane, Haversian/Volkmann's canal, and bone marrow. A Mann-Whitney U test was used for statistical processing, and p < .05 was considered to indicate a statistically significant difference. In the oral mucosa and the periodontal membrane, E+ had a significantly smaller vascular lumen area than E−. In the Haversian/Volkmann's canal and the bone marrow, no significant intergroup difference was observed in the intravascular lumen area. We postulate that this is due to a low smooth muscle content of blood vessels in the mandible and suggest that the vasoconstrictive effect of epinephrine-containing local anesthetics within the mandible is ineffective.

Background: Epithelial-mesenchymal transition (EMT) is considered to play a critical role in cancer progression and metastasis. However, the impact of EMT on the prognosis of hepatocellular carcinoma (HCC) is still elusive. In this study, we examined the relationship between the expression of EMT markers and recurrence-free survival (RFS) and overall survival (OS) in HCC patients after hepatic resection. Summary: The mRNA expression of 15 genes related to EMT was assessed by quantitative real-time polymerase chain reaction in cancerous tissues from 72 patients who underwent hepatic resection of HCC between January 2005 and December 2010 at our hospital. The upregulation of TWIST and the downregulation of tight junction protein ZO-1 (TJP1) were significantly associated with shorter RFS as well as OS. Increased levels of TWIST and decreased levels of TJP1 should be predictive markers for poor prognosis in patients with HCC after hepatectomy; those could serve as potential biomarkers for the treatment of HCC. Key Messages: A low level of TJP1 and high level of TWIST expression were prognostic factors predicting HCC after hepatic resection.

Whole genome-scale integrated analyses of exon array and array-comparative genomic hybridization are expected to enable the identification of unknown genetic features of cancer cells. Here, we evaluated this approach in 22 gastric and colorectal cancer cell lines, focusing on protein kinase genes and genes belonging to the cadherin-catenin family. Regarding alternative splicing patterns, several cancer cell lines predominantly expressed isoform 1 of protein kinase A catalytic subunit beta (PRKACB). Paired gastric cancer specimens demonstrated that isoform 1 of PRKACB was a novel cancer-related variant transcript in gastric cancers. In addition, the exon array analysis clearly identified exon 3 or exon 3-4 skipping in catenin beta 1, a short intron insertion with exon 9 skipping in CDH1, and a deletional transcript of CDH13. These abnormal transcripts were shown to have arisen from small genomic deletions. Meanwhile, an integrated analysis of 11 gastric cancer cell lines revealed that four cell lines amplified fibroblast growth factor receptor 2, with truncated forms observed in two of the cell lines. Gene amplification, and not the truncated form, was found to determine the sensitivity to a fibroblast growth factor receptor inhibitor, indicating that our cell line panel might be useful for cell-based evaluations of specific inhibitors. Using an integrated analysis, we identified several abnormal transcripts and genomic alterations in gastric and colorectal cancer cells. Our approach might enable genetic changes to be identified more efficiently, and the present results warrant further investigation using clinical samples and integrated analyses.

We describe a 19-year-old woman with onset of epileptic seizure, and a small mural nodule and multicystic lesions with severe brain edema located in the right frontal lobe. At surgery, the tumor and a clear margin was removed, and symptoms improved postoperatively. Extended local radiotherapy (60 Gy) was performed. Histopathological examination revealed oligodendroglioma-like tumor cells with a perinuclear halo. The tumor cells extended processes toward CD34-positive proliferating vessels, which resemble a basement membrane. These proliferating vessels formed a tumor membrane so that there was a clear margin between the tumor and brain tissue. Tumor cells were positive for epithelial membrane antigen in a dot-like pattern. MIB-1 staining index was 50.6%. Electron microscopy showed cilia and zipper-like junctions, and anaplastic clear-cell ependymoma grade III was diagnosed. A characteristic of the case was formation of a tumor membrane by proliferating tumor blood vessels. Fluorescence in situ hybridization showed 1p/19q deletions, and the concentration of erythropoietin in the cyst fluid was abnormally high, at 1,859.4 mIU/ml. Erythropoietin and erythropoietin receptors were verified with immunohistochemical staining.

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.

Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR.

The effect of motor cortex stimulation (MCS) therapy for deafferentation pain was evaluated based on c-Fos, a known pain marker. Nineteen mature cats weighing 1.5–3.5 kg were used. Cats were divided into three groups: a deafferentation pain group in which the left trigeminal ganglion was destroyed, an MCS group in which MCS was used following destruction of the trigeminal ganglion, and a control group. Sites and levels of c-Fos expression were examined immunohistochemically. The percentage of c-Fos-positive cells in the left spinal nucleus of the trigeminus, the bilateral insula, and the bilateral operculum increased in both the deafferentation pain and the MCS groups. There were no statistically significant differences between these groups. In the cingulate gyrus, the percentage of c-Fos-positive cells increased bilaterally in the deafferentation pain group and the MCS group, but the increase was greater in the MCS group. The increase in c-Fos-positive cells in the left spinal nucleus of the trigeminus in the deafferentation group may reflect reported electrical hyperactivity. The cingulate gyrus, insula, and parietal operculum were activated after deafferentation. This change (increase in c-Fos positive cells) is related to the development of deafferentation pain. Pain relief due to MCS is not dependent on the suppression of the activated left spinal nucleus of the trigeminus or the descending analgesic mechanism of the brain stem. Activation of the cingulate gyrus appears to be a factor in the analgesic mechanism of MCS.

Chronic subdural hematoma (CSH) is a common disease that is treated with burr hole drainage by neurosurgeons. The outcome of CSH is mostly favorable. We treated 2 cases with bilateral occipital lobe infarction due to CSH. A 57-year-old woman was ambulatory when she visited a clinic for evaluation of headache. One hour after the CT was taken, she developed acute impairment of consciousness, so that she was referred to our hospital. The second patient was a 73-year-old woman with a history of depression who was involved in a traffic accident 5 weeks before admission to our hospital. She was at first admitted to a psychiatric hospital for evaluation of gait disturbance. Three weeks after she was admitted to the psychiatric hospital, she fell into a coma. She was referred to our hospital. Their brain CT on admission revealed compressed ambient and interpeduncular cistern and bilateral CSH. Although burr hole drainage surgery was performed, the 2 patients developed severe sequelae due to occipital lobe infarction caused by central transtentorial herniation.

Two cases of spontaneous rupture of cystic craniopharyngioma without chemical meningitis are described. A 70-year-old woman complained of headache and visual field disturbance in July 1993. The tumor was extirpated in November 1993 and again in December 1996. After regular periodic follow-up evaluation, she was hospitalized for reoperation because of expansion of the cyst on magnetic resonance (MR) imaging in November 1998. However, preoperative MR imaging taken 8 weeks later revealed spontaneous reduction of the cyst. A 69-year-old woman noticed deteriorating vision and bitemporal hemianopsia in November 1998. The tumor was removed in December 1998, and 50.4 Gy postoperative radiotherapy was administered. MR imaging in May 2000 demonstrated an enlargement of the cyst, so she was hospitalized again for operation. However, preoperative MR imaging taken 7 weeks later showed spontaneous reduction of the cyst. Neither of the cases of cyst rupture were accompanied by symptoms of chemical meningitis. The signal intensity of the tumors on T1-weighted MR imaging declined after cyst reduction. Thereafter, the cysts increased in size again at 7 months and 5 months. Regular follow-up on MR imaging is necessary, since the cyst size can increase rapidly, even after spontaneous rupture.

SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.

Acquired resistance to antiangiogenic drugs has emerged as a potentially important issue in clinical settings; however, the underlying molecular and cellular mechanism of resistance to vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitor (TKI) remains largely unclear. We evaluated the cellular characteristics of human umbilical vein endothelial cell (HUVEC) clones, which are resistant to VEGFR2-TKI (Ki8751) to elucidate this mechanism of resistance to antiangiogenic drugs. Resistant HUVEC clones were 10-fold more resistant to VEGFR2-TKI than the parental cells and they exhibited an almost complete absence of VEGF-mediated cellular proliferation. The mRNA expression analysis revealed that expression of VEGFR1, VEGFR2 and VEGFR3 was lower in resistant clones, while that of several angiogenic ligands was increased. The protein expression of VEGFR2 was markedly down-regulated in two (R5 and R6 clone) out of five resistant clones. Focusing on the R5 clone, VEGF stimulation did not increase the phosphorylation of VEGFR2 or the dimerization of VEGFR2. The inhibition of phospho-AKT by VEGFR2-TKI was also weakened more than 10-fold in the R5 clone. Finally, a microarray analysis revealed that some angiogenesis-associated, and some angiogenesis-specific genes, including platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31, homeobox A9 (HOXA9), and endothelial cell-specific molecule 1 (ESM1), were remarkably down-regulated in all the resistant clones compared with the parental cells. HUVEC clones resistant to VEGFR2-TKI exhibited down-regulation of VEGFR2, a decreased signal response to VEGF stimulation, and the loss of vascular endothelial markers. These results strongly suggest that an escape from VEGFR2 signaling-dependency is one of the cellular mechanisms of resistance to VEGFR2-TKI in vascular endothelial cells.

Resveratrol (3, 4′, 5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and wine, has anti-proliferative activity on human-derived cancer cells. In our study, we used a conventional condensation reaction between aldehydes and amines to provide a number of aza-resveratrol (3, 4′, 5-trihydroxy-trans- aza-stilbene) derivatives in an attempt to screen for compounds with resveratrol’s action but with increased potency. Aza-resveratrol and its hydroxylated derivative (3, 4, 4′, 5-tetrahydroxy-trans- aza-stilbene) showed a more enhanced anti-proliferative effect than resveratrol in an MCF-7 breast carcinoma cell line. To identify the cellular targets of the aza derivatives of resveratrol, we conjugated the latter aza-stilbene compound with epoxy-activated agarose and performed affinity purification. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was identified as a major target protein in MCF-7 cell lysates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). The aza-resveratrol and its hydroxylated derivative, but not resveratrol, were also found to be potent inhibitors of MIF tautomerase activity, which may be associated with their inhibitory effects on MIF bioactivity for cell growth.

PURPOSE. The aim of this study was to investigate the expression changes of epithelial mesenchymal transition (EMT)-related molecules induced by TGF- signaling in a human corneal epithelial cell line (HCECs). METHODS. The cellular response to TGF- was evaluated by immunoblotting, quantitative real-time RT-PCR, and immunofluorescence microscopy in HCECs. RESULTS. TGF- significantly increased mRNA expression of SNAI1, SNAI2, VIM, and FN1, but not TWIST1 through Smad and non-Smad pathways in HCECs. Protein expression of a mesenchymal marker N-cadherin was dose-dependently increased and that of an epithelial marker of E-cadherin was decreased by TGF-. TGF-, but not EGF, mediated the EMTlike morphologic changes. Both TGF- and EGF were capable of upregulating SNAI1 and SNAI2 by about two-fold within a short response time. However, a detailed time course analysis revealed drastically different expression patterns, with TGFmediating a sustained upregulation of SNAI1 and SNAI2 for at least for 6 days and EGF allowing a return to the baseline expression values after 8 12 h. These data indicate that TGF-, but not EGF, induces sustained upregulation of SNAI1 and SNAI2 in HCECs. CONCLUSIONS. TGF- induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways, EMT-like morphologic changes, downregulation of E-cadherin, and upregulation of N-cadherin in HCECs. The authors’ findings provide insight into the TGF- signaling and the temporal expression patterns of EMT-inducible transcription factors in HCECs. (Invest Ophthalmol Vis Sci. 2011;52:2437‐2443) DOI

Bortezomib, a selective 26S proteasome inhibitor, has shown clinical benefits against refractory multiple myeloma. The indirect anti-angiogenic activity of bortezomib has been widely recognized; however, the growth-inhibitory mechanism of bortezomib on vascular endothelial cells remains unclear, especially on the cell cycle. Here, we showed that bortezomib (2 nM of the IC(50) value) potently inhibited the cellular growth of human umbilical vascular endothelial cells (HUVECs) via a vascular endothelial growth factor receptor (VEGFR)-independent mechanism resulting in the induction of apoptosis. Bortezomib significantly increased the vascular permeability of HUVECs, whereas a VEGFR-2 tyrosine kinase inhibitor decreased it. Interestingly, a cell cycle analysis using flow cytometry, the immunostaining of phospho-histone H3, and Giemsa staining revealed that bortezomib suppressed the G2/M transition of HUVECs, whereas the mitotic inhibitor paclitaxel induced M-phase accumulation. A further analysis of cell cycle-related proteins revealed that bortezomib increased the expression levels of cyclin B1, the cdc2/cyclin B complex, and the phosphorylation of all T14, Y15, and T161 residues on cdc2. Bortezomib also increased the ubiquitination of cyclin B1 and wee1, but inhibited the kinase activity of the cdc2/cyclin B complex. These protein modifications support the concept that bortezomib suppresses the G2/M transition, rather than causing M-phase arrest. In conclusion, we demonstrated that bortezomib potently inhibits cell growth by suppressing the G2/M transition, modifying G2/M-phase-related cycle regulators, and increasing the vascular permeability of vascular endothelial cells. Our findings reveal a cell cycle-related mode of action and strongly suggest that bortezomib exerts an additional unique vascular disrupting effect as a vascular targeting drug.

Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21CIP1/WAF1 expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 ± 301 versus 1735 ± 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family. Cancer Res; 70(5); 2053–63

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]

DelE746_A750-type EGFR is a constitutively active type of mutation that enhances EGFR signaling. However, the changes in gene expression that occur in mutant EGFR-harboring cells has not been fully studied.A gene expression analysis of HEK293 cells transfected with wild-type or mutant EGFR was performed focusing on the significant gene.Early growth response 1 (EGR1), a transcription factor, was the most strongly up-regulated gene in mutant EGFR-transfected cells among the genes examined. An increase in EGR1 expression in the mutant EGFR cells was confirmed using RT-PCR or immunoblotting. The expression was up-regulated by EGF stimulation and down-regulated by EGFR-tyrosine kinase inhibitor. In addition, the MEK inhibitor U0126 inhibited EGR1 expression, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not.Mutant EGFR constitutively up-regulates EGR1 through the ERK pathway, and its expression is correlated with EGFR signal activation. Findings provide an insight into a target gene of mutant EGFR and further improve the understanding of the oncogenic properties of EGFR.

SRPX2 (Sushi repeat containing protein, X-linked 2) was first identified as a downstream molecule of the E2A-HLF fusion gene in t(17;19)-positive leukemia cells and the biological function of this gene remains unknown. We found that SRPX2 is overexpressed in gastric cancer and the expression and clinical features showed that high mRNA expression levels were observed in patients with unfavorable outcomes using real-time RT-PCR. The cellular distribution of SRPX2 protein showed the secretion of SRPX2 into extracellular regions and its localization in the cytoplasm. The introduction of the SRPX2 gene into HEK293 cells did not modulate the cellular proliferative activity but did enhance the cellular migration activity, as shown using migration and scratch assays. The conditioned-medium obtained from SRPX2-overexpressing cells increased the cellular migration activity of a gastric cancer cell line, SNU-16. In addition, SRPX2 protein remarkably enhanced the cellular adhesion of SNU-16 and HSC-39 and increased the phosphorylation levels of focal adhesion kinase (FAK), as shown using western blotting, suggesting that SRPX2 enhances cellular migration and adhesion through FAK signaling. In conclusion, the overexpression of SRPX2 enhances cellular migration and adhesion in gastric cancer cells. Here, we report that the biological functions of SRPX2 include cellular migration and adhesion to cancer cells. © 2008 Wiley-Liss, Inc.

Two cases of spontaneous rupture of cystic craniopharyngioma without chemical meningitis are described. A 70-year-old woman complained of headache and visual field disturbance in July 1993. The tumor was extirpated in November 1993 and again in December 1996. After regular periodic follow-up evaluation, she was hospitalized for reoperation because of expansion of the cyst on magnetic resonance (MR) imaging in November 1998. However, preoperative MR imaging taken 8 weeks later revealed spontaneous reduction of the cyst. A 69-year-old woman noticed deteriorating vision and bitemporal hemianopsia in November 1998. The tumor was removed in December 1998, and 50.4 Gy postoperative radiotherapy was administered. MR imaging in May 2000 demonstrated an enlargement of the cyst, so she was hospitalized again for operation. However, preoperative MR imaging taken 7 weeks later showed spontaneous reduction of the cyst. Neither of the cases of cyst rupture were accompanied by symptoms of chemical meningitis. The signal intensity of the tumors on T1-weighted MR imaging declined after cyst reduction. Thereafter, the cysts increased in size again at 7 months and 5 months. Regular follow-up on MR imaging is necessary, since the cyst size can increase rapidly, even after spontaneous rupture.

We report successful operations for a meningeal hemangiopericytoma using sufficient amounts of Preoperative Autologous Transfusion (PAT) and Hemodilutional Autologous Transfusion (HAT). A 23-year-old woman with amenorrhea and bilateral visual field disturbance was found to have a huge intracranial tumor. MRI showed a well-enhanced cystic mass in the left middle fossa, suprasellar, intrasellar, sphenoidal sinus, and cavernous sinus. Preoperatively, the tumor was thought to be a cystic pituitary tumor or meningioma. Surgical removal was planned in three steps. The first operation was carried out via the transsphenoidal approach. Total blood loss was 1348 ml and 2 MAP infusion were required to control bleeding. Histopathological diagnosis was hemangiopericytoma. After preparation of PAT 400 ml and HAT 800 ml, we carried out the second partial removal operation mainly via the interhemispheric approach. Total blood loss was 1829 ml and required autologous transfusion only. After preparation of PAT 1200 ml and HAT 400 ml, the last total removal operation was carried out mainly via the pterional and subtemporal approach. Total blood loss was 1813 ml and required autologous transfusion only. We needed 2 MAP infusion in the first operation, but were able to perform total removal successfully without homologous blood transfusion because a sufficient amount of PAT and HAT had been prepared preoperatively. Hemangiopericytoma required postoperative radiation therapy to avoid local recurrence. After successful removal of the tumor surgically, postoperative radiation therapy was able to be carried out efficiently.

Epithelial-Mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. However, whether the overexpression of EMT-related genes mediates the malignant phenotype in HCC remains largely unclear. In this study, we examined the relationship between mRNA expression levels of EMT-related genesand 12 clinical characteristics including recurrence-free survival and overall survival in HCC patients who had undergone curative hepatic resection. The mRNA expression levels of 15 genes (CDH1, CDH2, Snail, Slug, ZEB1, ZEB2, TWIST, Vimentin, Fibronectin, DDR2, S100A4, TJP-1/ZO-1, FOXC2, SIX1, GSC) was determined by quantitative real-time PCR in tissues from 72 HCC tumors. Twist expression was significantly upregulated in recurrence group (p=0.02). The upregulation of Twist and the downregulation of TJP1 were significantly related with poor prognosis in univariate and multivariate analysis. In conclusion, Twist and TJP1 may be predictive biomarkers in patients with HCC after hepatectomy. Our findings provide a novel insight into TJP1 and HCC, warranting further investigations. 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 3406. doi:1538-7445.AM2012-3406

Objectives: A number of antiangiogenic inhibitors have been studied in clinical settings, some of which are now under clinical evaluation in pancreas cancers (PC), therefore, prognostic marker of angiogenesis-related molecules in PC is needed. The aim of this study is to evaluate the serum levels of angiogenesis-related factors in patients with pancreas cancer. Methods: The plasma samples obtained from 45 pancreas cancers (PC) were analyzed compared with 9 pancreatitis (P), 16 benign hepatobiliary diseases (HD) and 58 colorectal cancers (CC). Serum angiogenesis-related factors including Angiopoietin-2, Follistatin, G-CSF, HGF, IL-8, Leptin, PDGF-BB, PECAM-1 and VEGF were determined by antibody suspension bead arrays system. Results: All of the serum angiogenesis-related factors were not increased in PC compared with P and HD, while those of CC were markedly increased. Although IL-8 levels were not increased in most samples of PC, it was significantly increased in patients with distant metastases and there was a trend toward to a shorter survival. Conclusions: Serum levels of angiogenesis-related factors were basically not increased in patients with PC, however, serum IL-8 levels may reflect PC progression and shorter survival. 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 4185. doi:10.1158/1538-7445.AM2011-4185

Epithelial-Mesenchymal Transition (EMT) has been recognized as a cellular process by which epithelial cells undergo mesenchymal phenotype leading to increased motility and invasion in cancer cells. We recently have reported that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in hepatocellular carcinoma (HCC). Large-scale clinical data on SNAI1 expression and the prognosis of patients with HCC was recently reported, and the overexpression of Snail and/or Twist was correlated with a worse prognosis. However, it remains largely unclear on clinico-pathological features of other EMT-related genes and whether these genes are overexpressed in HCC compared with paired non-cancer lesion of the liver. Here, we examined the mRNA expression levels of EMT-related genes including SNAI1/Snail, SNAI2/slug, ZEB1/TCF8, ZEB2/SIP1, TWIST, CDH1/E-cadherin, CDH2/N-cadherin, VIM/vimentin and FN1/fibronectin in HCC. RNA was extracted from frozen surgical HCC samples (n=48) and their paired liver samples (n=14). Real-time RT-PCR amplification was performed. Glyceraldehyde 3-phosphate dehydrogenase (GAPD) was used to normalize the expression levels in the subsequent quantitative analyses. E-cadherin was significantly downregulated in most of HCC (12/14) and those of 5 HCC were markedly downregulated below 25% of paired liver. Interestingly, we found that ZEB1 is tended to be upregulated in HCC, while ZEB2 was downregulated. Regarding EMT-inducible transcription factors, SNAI1, SNAI2, ZEB1, ZEB2 and TWIST, these expression levels were overexpressed in several HCC samples. In conclusion, our findings indicate that some HCC subpopulation actually undergo EMT via upregulation of SNAI1, SNAI2, ZEB1, ZEB2 and TWIST. Evaluation of clinico-pathological features on these expression and analysis of additional HCC samples up to 100 cases are now under way. 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 3371. doi:10.1158/1538-7445.AM2011-3371

Activation of the HGF-MET signaling increases invasion, metastasis and angiogenesis in cancer cells. Therefore, HGF-MET signaling is regarded as an oncogenic signaling pathway, and intensive therapeutic approaches focusing on this signaling pathway are ongoing in the field of cancer treatment. We previously reported that the concentration of serum HGF was strongly related with the treatment outcome of EGFR-tyrosine kinase inhibitors in patients with adenocarcinoma of the lung (Kasahara K. et al, Clin Cancer Res. 2010). Meanwhile, recent study has clearly demonstrated that HGF expression was regulated by short poly A sequence in its promoter region in breast cancer cells (Ma J. et al, J Clin Invest. 2009). In this study, we evaluated the poly A sequence in HGF promoter region and expression levels of HGF in 51 cancer cell lines. The five cell lines exhibited a short poly A sequence (16A-25A) in HGF promoter compared with wild-type (30A). Two of five cell lines overexpressed HGF at mRNA and protein expression levels determined by quantitative RT-PCR and ELISA. On the other hand, there were no HGF-overexpressing cell lines among 46 cell lines with wild-type poly A sequence. Of note, we have also detected the short poly A sequence in cancer cells in patients with lung cancer. Evaluation of short poly A, HGF expression and treatment outcomes of EGFR-tyrosine kinase inhibitors is under way. 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 1095. doi:10.1158/1538-7445.AM2011-1095

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. We recently have reported that the expression of CD133 in cancer cells could be regulated though the inhibition of signal transduction by molecular-targeted drugs (Matsumoto K. et al. Cancer Res. 2009). Here, we screened the expression changes of CD133 using wide variety of molecular-targeted drugs in FGFR2-amplified and CD133-overexpressing gastric cancer cell lines. CD133 expression was remarkably upregulated by FGFR inhibitor PD173074 exposure in these cells. Further analysis revealed that PD173074 upregulated the CD133 expression both mRNA and protein levels in dose- and time-dependent manners. In addition, the expression change was correlated with the mRNA expression levels of stem cell factors including OCT3/4, SOX2 and NANOG. As expected, PD173074 exhibited a potent growth inhibitory effect on FGFR2-amplified gastric cancer cells in vitro. These results suggest that the mechanism regulating the expression of CD133 is involved in FGFR2 signaling and also in addicted signaling pathway for cancer cells in FGFR2-amplified gastric 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 5207. doi:10.1158/1538-7445.AM2011-5207

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

Resveratrol (3, 4’, 5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and wine, has an anti-proliferative activity in human-derived cancer cells. In our study, we used a conventional condensation reaction between aldehydes and amines to provide a number of aza-resveratrol (3, 4’, 5-trihydroxy-trans- aza-stilbene) derivatives in an attempt to screen for compounds with resveratrol's action but with increased potency. Aza-resveratrol and its hydroxylated derivative (3, 4, 4’, 5-tetrahydroxy-trans- aza-stilbene) showed a more enhanced anti-proliferative effect than resveratrol in an MCF-7 breast carcinoma cell line. To identify the cellular targets of the aza derivatives of resveratrol, we conjugated the latter aza-stilbene compound with epoxy-activated agarose and performed an affinity purification. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was identified as a major target protein in MCF-7 cell lysates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). The aza-resveratrol and its hydroxylated derivative, but not resveratrol, were also found to be potent inhibitors of MIF tautomerase activity, which may be associated with their inhibitory effects on MIF bioactivity for cell growth. Such inhibitors may lead to the development of drugs for the treatment of cancer and, possibly, inflammatory and autoimmune diseases. 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 1099. doi:10.1158/1538-7445.AM2011-1099

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.

Forkhead Box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (28-fold / colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using siRNA resulted in a decrease in p21CIP1/WAF1 expression, and a reporter assay and ChIP assay showed that p21CIP1/WAF1 was one of target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21CIP1/WAF1 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity (EGFP: 2/15, FOXQ1: 7/15) and enhanced tumor growth (437 ± 301 vs. 1735 ± 769 mm3, p In conclusion, FOXQ1 is overexpressed in colorectal cancer and enhances tumorigenicity and tumor growth presumably through its angiogenic and anti-apoptotic effects. Our findings demonstrate that FOXQ1 is a new member of the cancer-related FOX family. 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 3893.

Epidermal growth factor receptor (EGFR) is often highly expressed in many solid tumors and EGFR tyrosine kinase inhibitor (EGFR-TKI) demonstrated the therapeutic benefit for non small cell lung cancer in clinical setting. We previously established the EGFR-TKI resistant cell lines derived from highly parental sensitive cell line with constitutively active EGFR mutation, PC-9. EGFR-TKI-resistant cell line shows about 1000 fold resistant for EGFR-TKI compared with parental PC-9 cells. We performed the microarray and exon array analysis for these cell lines and identified that 30 genes were altered the gene expressions over 5 fold between two cell lines. There were no gene expression changes in EGFR, HER4, AKT and adaptor molecules, whereas HER2 and HER3 were down-regulated and MET were up-regulated in resistant cells. Interestingly, several ligands of EGFR such as TGF alpha, epiregulin, amphiregulin and HB-EGF were up-regulated in EGFR-TKI resistant cell lines, suggesting that the ligand expression change are associated with the phenotype of drug resistant and may be useful molecular biomarkers for EGFR-TKI treatment. We are now investigating the functional analysis for these identified genes and analysing the result of exon array. 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 3994.

Acquired resistance to anti-angiogenetic drugs have emerged as a potentially important problem in clinical settings, however, the underlying mechanism remains largely unclear. In this study, we established human umbilical vein endothelial cell (HUVEC) clones that are resistant to VEGFR tyrosine kinase inhibitor (VEGFR-TKI, Ki8751) using MNNG and VEGFR-TKI. In growth inhibition assay, VEGFR-TKI resistant HUVEC clones were significantly decreased cellular sensitivity to Ki8751 about 4-10 fold compared with that in the parental cells. VEGF-mediated cellular proliferations of resistant clones were also decreased. Interestingly, VEGFR2 expression was down-regulated and the phosphorylation levels of Akt were up-regulated in resistant clones. Finally, microarray analysis revealed that several angiogenesis-related or -specific genes including CD31 were remarkably down-regulated in resistant clones compared with the parental cells. In conclusion, these results suggest that the mechanism of VEGFR-TKI resistant is closely involved in “escape phenomenon” from VEGF-VEGFR system and our findings provide a novel insight into the drug resistant to VEGFR-TKI in vascular endothelial 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 370.

Alternative splicing is a major source of protein diversity and cancer is driven by mutations in genes that control the proliferation of cells. The specific exon abnormality may contribute to the etiology of cancer and may provide selective drug targets. However, most studies of alternative splicing in human disease have used a targeted approach and focused on individual genes among whole genome. In this study, we evaluated an integrated approach of whole genome scale exon array and SNP array to detect cancer-associated alternative splicing, especially focusing on the protein kinase family. We investigated exon-level expression profiling against 22 gastric and colorectal cancer cell lines using Affymetrix Human Exon 1.0 ST Array. Array-based comparative genomic hybridization (CGH) analysis using high-density SNP Array (Affymetrix 250K Nsp /SNP6.0 Array) was also performed to explore genomic amplification/deletion. Several candidate alternative splicing or abnormalities of specific exon expression were detected in cancer-associated genes which detected genomic amplification/deletion frequently; exampling FGFR2, CDKN2A, and CDH13. We found the gene amplification of FGFR2 with C-terminus truncation. We also detected the existence of short CDH13 transcript resulted from small genomic deletion including consecutive 3 exons. In addition, exon-level expression pattern was compared between gastric and colorectal cancer cell lines. In conclusion, integration of whole genome exon array analysis and array CGH analysis may be a promising approach to explore the alternative exon abnormalities. 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 2156.

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.