Hiroyasu Kaneda

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 p21(CIP1/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.

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-1 alpha (HIF-1 alpha), a downstream molecule in the mTOR signaling pathway, might regulate CD133 expression; we therefore investigated the relation between CD133 and HIF-1 alpha. Hypoxic conditions up-regulated HIF-1 alpha expression and inversely down-regulated CD133 expression at both the mRNA and protein levels. Similarly, the HIF-1 alpha 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-1 alpha and HIF-1 beta were examined using clinical gastric cancer samples. A strong inverse correlation (r = -0.68) was observed between CD133 and HIF-1 alpha, but not between CD133 and HIF-1 beta. In conclusion, these results indicate that HIF-1 alpha 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-1 alpha 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.

Activation of HER family signaling as a mechanism of acquired resistance to ALK inhibitors in EML4-ALK-positive non-small cell lung cancer

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non–small cell lung cancer positive for the echinoderm microtubule-associated protein–like 4 (EML4)–ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown. Experimental Design: We established lines of EML4-ALK–positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK–positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics. Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal–regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion. Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK. Clin Cancer Res; 18(22); 6219–26. ©2012 AACR.

Phase I Safety, Pharmacokinetic, and Biomarker Study of BIBF 1120, an Oral Triple Tyrosine Kinase Inhibitor in Patients with Advanced Solid Tumors

BIBF 1120 is an oral multitargeted tyrosine kinase inhibitor that blocks the activity of vascular endothelial growth factor (VEGF) and other growth factor receptors. We have done a phase I study to evaluate the safety, pharmacokinetics, and pharmacodynamic biomarkers of BIBF 1120. Patients with advanced refractory solid tumors were treated with BIBF 1120 at oral doses of 150 to 250 mg twice daily. Drug safety and pharmacokinetics were evaluated, as were baseline and post-treatment levels of circulating CD117-positive bone marrow–derived progenitor cells and plasma soluble VEGF receptor 2 as potential biomarkers for BIBF 1120. Twenty-one patients were treated at BIBF 1120 doses of 150 (n = 3), 200 (n = 12), or 250 mg twice daily (n = 6). Dose-limiting toxicities of reversible grade 3 or 4 elevations of liver enzymes occurred in 3 of 12 patients at 200 mg twice daily and 3 of 6 patients at 250 mg twice daily. Stable disease was achieved in 16 (76.2%) patients, and median progression-free survival was 113 days (95% confidence interval, 77-119 d). Pharmacokinetic analysis indicated that the maximum plasma concentration and area under the curve for BIBF 1120 increased with the dose within the dose range tested. Levels of CD117-positive bone marrow–derived progenitors and soluble VEGF receptor 2 decreased significantly during treatment over all BIBF 1120 dose cohorts. In conclusion, the maximum tolerated dose of BIBF 1120 in the current study was determined to be 200 mg twice daily, and our biomarker analysis indicated that this angiokinase inhibitor is biologically active. Mol Cancer Ther; 9(10); 2825–33. ©2010 AACR.

Sorafenib Inhibits the Hepatocyte Growth Factor–Mediated Epithelial Mesenchymal Transition in Hepatocellular Carcinoma

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.

Prophylactic cranial irradiation versus observation in patients with extensive-disease small-cell lung cancer: a multicentre, randomised, open-label, phase 3 trial

BACKGROUND Results from a previous phase 3 study suggested that prophylactic cranial irradiation reduces the incidence of symptomatic brain metastases and prolongs overall survival compared with no prophylactic cranial irradiation in patients with extensive-disease small-cell lung cancer. However, because of the absence of brain imaging before enrolment and variations in chemotherapeutic regimens and irradiation doses, concerns have been raised about these findings. We did a phase 3 trial to reassess the efficacy of prophylactic cranial irradiation in the treatment of extensive-disease small-cell lung cancer. METHODS We did this randomised, open-label, phase 3 study at 47 institutions in Japan. Patients with extensive-disease small-cell lung cancer who had any response to platinum-based doublet chemotherapy and no brain metastases on MRI were randomly assigned (1:1) to receive prophylactic cranial irradiation (25 Gy in ten daily fractions of 2·5 Gy) or observation. All patients were required to have brain MRI at 3-month intervals up to 12 months and at 18 and 24 months after enrolment. Randomisation was done by computer-generated allocation sequence, with age as a stratification factor and minimisation by institution, Eastern Cooperative Oncology Group performance status, and response to initial chemotherapy. The primary endpoint was overall survival, analysed in the intention-to-treat population. This trial is registered with the UMIN Clinical Trials Registry, number UMIN000001755, and is closed to new participants. FINDINGS Between April 3, 2009, and July 17, 2013, 224 patients were enrolled and randomly assigned (113 to prophylactic cranial irradiation and 111 to observation). In the planned interim analysis on June 18, 2013, of the first 163 enrolled patients, Bayesian predictive probability of prophylactic cranial irradiation being superior to observation was 0·011%, resulting in early termination of the study because of futility. In the final analysis, median overall survival was 11·6 months (95% CI 9·5-13·3) in the prophylactic cranial irradiation group and 13·7 months (10·2-16·4) in the observation group (hazard ratio 1·27, 95% CI 0·96-1·68; p=0·094). The most frequent grade 3 or worse adverse events at 3 months were anorexia (six [6%] of 106 in the prophylactic cranial irradiation group vs two [2%] of 111 in the observation group), malaise (three [3%] vs one [<1%]), and muscle weakness in a lower limb (one [<1%] vs six [5%]). No treatment-related deaths occurred in either group. INTERPRETATION In this Japanese trial, prophylactic cranial irradiation did not result in longer overall survival compared with observation in patients with extensive-disease small-cell lung cancer. Prophylactic cranial irradiation is therefore not essential for patients with extensive-disease small-cell lung cancer with any response to initial chemotherapy and a confirmed absence of brain metastases when patients receive periodic MRI examination during follow-up. FUNDING The Ministry of Health, Labour and Welfare of Japan.

SRPX2 is overexpressed in gastric cancer and promotes cellular migration and adhesion.

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.

Risk Factors for Cisplatin-Induced Nephrotoxicity and Potential of Magnesium Supplementation for Renal Protection

Background Nephrotoxicity remains a problem for patients who receive cisplatin chemotherapy. We retrospectively evaluated potential risk factors for cisplatin-induced nephrotoxicity as well as the potential impact of intravenous magnesium supplementation on such toxicity. Patients and Methods We reviewed clinical data for 401 patients who underwent chemotherapy including a high dose (≥60 mg/m2) of cisplatin in the first-line setting. Nephrotoxicity was defined as an increase in the serum creatinine concentration of at least grade 2 during the first course of cisplatin chemotherapy, as assessed on the basis of National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. The severity of nephrotoxicity was evaluated on the basis of the mean change in the serum creatinine level. Magnesium was administered intravenously to 67 patients (17%). Results Cisplatin-induced nephrotoxicity was observed in 127 patients (32%). Multivariable analysis revealed that an Eastern Cooperative Oncology Group performance status of 2 (risk ratio, 1.876; P = 0.004) and the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) (risk ratio, 1.357; P = 0.047) were significantly associated with an increased risk for cisplatin nephrotoxicity, whereas intravenous magnesium supplementation was associated with a significantly reduced risk for such toxicity (risk ratio, 0.175; P = 0.0004). The development of hypomagnesemia during cisplatin treatment was significantly associated with a greater increase in serum creatinine level (P = 0.0025). Magnesium supplementation therapy was also associated with a significantly reduced severity of renal toxicity (P = 0.012). Conclusions A relatively poor performance status and the regular use of NSAIDs were significantly associated with cisplatin-induced nephrotoxicity, although the latter association was marginal. Our findings also suggest that the ability of magnesium supplementation to protect against the renal toxicity of cisplatin warrants further investigation in a prospective trial.

Activin A inhibits vascular endothelial cell growth and suppresses tumour angiogenesis in gastric cancer.

Background:Activin A is a multi-functional cytokine belonging to the transforming growth factor-β (TGF-β) superfamily; however, the effect of activin A on angiogenesis remains largely unclear. We found that inhibin β A subunit (INHBA) mRNA is overexpressed in gastric cancer (GC) specimens and investigated the effect of activin A, a homodimer of INHBA, on angiogenesis in GC.Methods:Anti-angiogenic effects of activin A via p21 induction were evaluated using human umbilical vein endothelial cells (HUVECs) in vitro and a stable INHBA-introduced GC cell line in vivo.Results:Compared with TGF-β, activin A potently inhibited the cellular proliferation and tube formation of HUVECs with induction of p21. A promoter assay and a chromatin immunoprecipitation assay revealed that activin A directly regulates p21 transcriptional activity through Smads. Stable p21-knockdown significantly enhanced the cellular proliferation of HUVECs. Notably, stable p21-knockdown exhibited a resistance to activin-mediated growth inhibition in HUVECs, indicating that p21 induction has a key role on activin A-mediated growth inhibition in vascular endothelial cells. Finally, a stable INHBA-introduced GC cell line exhibited a decrease in tumour growth and angiogenesis in vivo.Conclusion:Our findings highlight the suppressive role of activin A, unlike TGF-β, on tumour growth and angiogenesis in GC.

Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non-small-cell lung cancer based on T790M status after disease progression during EGFR-TKI treatment

Background The efficacy of programmed death-1 blockade in epidermal growth factor receptor gene (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) patients with different mechanisms of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) is unknown. We retrospectively evaluated nivolumab efficacy and immune-related factors in such patients according to their status for the T790M resistance mutation of EGFR. Patients and methods We identified 25 patients with EGFR mutation-positive NSCLC who were treated with nivolumab after disease progression during EGFR-TKI treatment (cohort A). Programmed death-ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) density in tumor specimens obtained after acquisition of EGFR-TKI resistance were determined by immunohistochemistry. Whole-exome sequencing of tumor DNA was carried out to identify gene alterations. The relation of T790M status to PD-L1 expression or TIL density was also examined in an independent cohort of 60 patients (cohort B). Results In cohort A, median progression-free survival (PFS) was 2.1 and 1.3 months for T790M-negative and T790M-positive patients, respectively (P = 0.099; hazard ratio of 0.48 with a 95% confidence interval of 0.20-1.24). Median PFS was 2.1 and 1.3 months for patients with a PD-L1 expression level of ≥1% or <1%, respectively (P = 0.084; hazard ratio of 0.37, 95% confidence interval of 0.10-1.21). PFS tended to increase as the PD-L1 expression level increased with cutoff values of ≥10% and ≥50%. The proportion of tumors with a PD-L1 level of ≥10% or ≥50% was higher among T790M-negative patients than among T790M-positive patients of both cohorts A and B. Nivolumab responders had a significantly higher CD8+ TIL density and nonsynonymous mutation burden. Conclusion T790M-negative patients with EGFR mutation-positive NSCLC are more likely to benefit from nivolumab after EGFR-TKI treatment, possibly as a result of a higher PD-L1 expression level, than are T790M-positive patients.