Description: Purpose: To determine the prognostic significance of miR-663 in glioblastoma, its effect in tumor progression, and the underlying mechanism. Experimental Design: Specimens from 256 cases of patients with glioma, including 239 patients with follow-up information, were used to analyze the association between miR-663 and patients' prognosis by Kaplan–Meier and multivariate Cox regression analyses. The effects of miR-663 on glioblastoma cell proliferation and invasion were examined both in vitro and in vivo . Bioinformatics prediction and signal network analysis were applied to identify the putative targets of miR-663, which were further verified by luciferase reporter assay, rescue experiments as well as the immunohistochemistry (IHC) and Western blotting examination of downstream effectors. Quantitative reverse transcriptase PCR (qRT-PCR) and IHC were applied to investigate the clinical association between miR-663 and its target in human glioblastoma specimens. Results: miR-663 was inversely correlated with glioma grades but positively correlated with patients' survival. Furthermore, two distinct subgroups of patients with glioblastoma with different prognoses were identified on the basis of miR-663 expression in our specimens and that from The Cancer Genome Atlas (TCGA) database. Overexpression of miR-663 significantly suppressed the proliferation and invasion of glioblastoma cells in vitro and in vivo . Mechanistically, we discovered PIK3CD as a direct target of miR-663 and found that phosphorylated AKT and three key downstream effectors of PIK3CD , i.e., CCND1, MMP2, and MMP7, were downregulated by miR-663 overexpression. Moreover, PIK3CD was inversely correlated with miR-663 in glioblastoma specimens and predicted poor prognosis of patients with glioblastoma. Conclusion: miR-663 is a novel prognostic biomarker and a potential therapeutic candidate for glioblastoma. Clin Cancer Res; 20(7); 1803–13. ©2014 AACR .

Description: Non–small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. Cancer Res; 75(20); 4372–83. ©2015 AACR.

Description: Purpose: Pazopanib is an effective treatment for advanced renal cell carcinoma and soft-tissue sarcoma. Transaminase elevations have been commonly observed in pazopanib-treated patients. We conducted pharmacogenetic analyses to explore mechanistic insight into pazopanib-induced liver injury. Experimental Design: The discovery analysis tested association between four-digit HLA alleles and alanine aminotransferase (ALT) elevation in pazopanib-treated patients with cancer from eight clinical trials ( N = 1,188). We conducted confirmatory analysis using an independent dataset of pazopanib-treated patients from 23 additional trials ( N = 1,002). Genome-wide association study (GWAS) for transaminase elevations was also conducted. Results: The discovery study identified an association between HLA-B *57:01 carriage and ALT elevation [ P = 5.0 x 10 –5 for maximum on-treatment ALT (MaxALT); P = 4.8 x 10 –4 for time to ALT 〉 3 x upper limit of normal (ULN) event; P = 4.1 x 10 –5 for time to ALT 〉 5 x ULN event] that is significant after adjustment for number of HLA alleles tested. We confirmed these associations with time to ALT elevation event ( P = 8.1 x 10 –4 for ALT 〉 3 x ULN, P = 9.8 x 10 –3 for ALT 〉 5 x ULN) in an independent dataset. In the combined data, HLA-B *57:01 carriage was associated with ALT elevation ( P = 4.3 x 10 –5 for MaxALT, P = 5.1 x 10 –6 for time to ALT 〉 3 x ULN event, P = 5.8 x 10 –6 for time to ALT 〉 5 x ULN event). In HLA-B *57:01 carriers and noncarriers, frequency of ALT 〉 3 x ULN was 31% and 19%, respectively, and frequency of ALT 〉 5 x ULN was 18% and 10%, respectively. GWAS revealed a possible borderline association, which requires further evaluation. Conclusions: These data indicate that HLA-B *57:01 carriage confers higher risk of ALT elevation in patients receiving pazopanib and provide novel insight implicating an immune-mediated mechanism for pazopanib-associated hepatotoxicity in some patients. Clin Cancer Res; 22(6); 1371–7. ©2015 AACR .

Description: Purpose: VEGF receptor (VEGFR) kinases are important drug targets in oncology that affect function of systemic endothelial cells. To discover genetic markers that affect VEGFR inhibitor pharmacodynamics, we performed a genome-wide association study of serum soluble vascular VEGFR2 concentrations [sVEGFR2], a pharmacodynamic biomarker for VEGFR2 inhibitors. Experimental Design: We conducted a genome-wide association study (GWAS) of [sVEGFR2] in 736 healthy Old Order Amish volunteers. Gene variants identified from the GWAS were genotyped serially in a cohort of 128 patients with advanced solid tumor with baseline [sVEGFR2] measurements, and in 121 patients with renal carcinoma with [sVEGFR2] measured before and during pazopanib therapy. Results: rs34231037 (C482R) in KDR , the gene encoding sVEGFR2 was found to be highly associated with [sVEGFR2], explaining 23% of the variance ( P = 2.7 x 10 –37 ). Association of rs34231037 with [sVEGFR2] was replicated in 128 patients with cancer with comparable effect size ( P = 0.025). Furthermore, rs34231037 was a significant predictor of changes in [sVEGFR2] in response to pazopanib ( P = 0.01). Conclusion: Our findings suggest that genome-wide analysis of phenotypes in healthy populations can expedite identification of candidate pharmacogenetic markers. Genotyping for germline variants in KDR may have clinical utility in identifying patients with cancer with unusual sensitivity to effects of VEGFR2 kinase inhibitors. Clin Cancer Res; 21(2); 365–72. ©2014 AACR .

Description: Genome-wide association studies (GWAS) of bladder cancer have identified a number of susceptibility loci in European populations but have yet to uncover the genetic determinants underlying bladder cancer incidence among other ethnicities. Therefore, we performed the first GWAS in a Chinese cohort comprising 3,406 cases of bladder cancer and 4,645 controls. We identified a new susceptibility locus for bladder cancer at 5q12.3, located in the intron of CWC27 (rs2042329), that was significantly associated with disease risk (OR = 1.40; P = 4.61 × 10−11). However, rs2042329 was not associated with bladder cancer risk in patients of European descent. The rs2042329 risk allele was also related to significantly increased expression levels of CWC27 mRNA and protein in bladder cancer tissues from Chinese patients. Additional functional analyses suggested that CWC27 played an oncogenic role in bladder cancer by inducing cell proliferation and suppressing apoptosis. In conclusion, the identification of a risk-associated locus at 5q12.3 provides new insights into the inherited susceptibility to bladder cancer in Chinese populations and may help to identify high-risk individuals. Cancer Res; 76(11); 3277–84. ©2016 AACR.

Description: The discovery of oncogenic driver mutations and the subsequent developments in targeted therapies have led to improved outcomes for subsets of lung cancer patients. The identification of additional oncogenic and drug-sensitive alterations may similarly lead to new therapeutic approaches for lung cancer. We identify and characterize novel FGFR2 extracellular domain insertion mutations and demonstrate that they are both oncogenic and sensitive to inhibition by FGFR kinase inhibitors. We demonstrate that the mechanism of FGFR2 activation and subsequent transformation is mediated by ligand-independent dimerization and activation of FGFR2 kinase activity. Both FGFR2-mutant forms are predominantly located in the endoplasmic reticulum and Golgi but nevertheless can activate downstream signaling pathways through their interactions with fibroblast growth factor receptor substrate 2 (FRS2). Our findings provide a rationale for therapeutically targeting this unique subset of FGFR2-mutant cancers as well as insight into their oncogenic mechanisms. Cancer Res; 75(15); 3139–46. ©2015 AACR.

Description: Non–small cell lung cancers (NSCLC) marked by EGFR mutations tend to develop resistance to therapeutic EGFR inhibitors, often due to secondary mutation EGFRT790M but also other mechanisms. Here we report support for a rationale to target IKBKE, an IκB kinase family member that activates the AKT and NF-κB pathways, as one strategy to address NSCLC resistant to EGFR inhibitors. While wild-type and mutant EGFR directly interacted with IKBKE, only mutant EGFR phosphorylated IKBKE on residues Y153 and Y179. The unphosphorylatable mutant IKBKE-Y153F/Y179-F that lost kinase activity failed to activate AKT and inhibited EGFR signaling. In clinical specimens of NSCLC with activating mutations of EGFR, we observed elevated levels of phospho-Y153 IKBKE. IKBKE ablation with shRNA or small-molecule inhibitor amlexanox selectively inhibited the viability of NSCLC cells with EGFR mutations in vitro. In parallel, we found that these treatments activated the MAPK pathway due to attenuation of an IKBKE feedback mechanism. In vivo studies revealed that combining amlexanox with MEK inhibitor AZD6244 significantly inhibited the xenograft tumor growth of NSCLC cells harboring activating EGFR mutations, including EGFRT790M. Overall, our findings define IKBKE as a direct effector target of EGFR and provide a therapeutic rationale to target IKBKE as a strategy to eradicate EGFR-TKI–resistant NSCLC cells. Cancer Res; 76(15); 4418–29. ©2016 AACR.

Description: Adoptive cell transfer (ACT) is considered a promising modality for cancer treatment, but despite ongoing improvements, many patients do not experience clinical benefits. The tumor microenvironment is an important limiting factor in immunotherapy that has not been addressed fully in ACT treatments. In this study, we report that upregualtion of the immunosuppressive receptor programmed cell death-1 (PD-1) expressed on transferred T cells at the tumor site, in a murine model of ACT, compared with its expression on transferred T cells present in the peripheral blood and spleen. As PD-1 can attenuate T-cell–mediated antitumor responses, we tested whether its blockade with an anti–PD-1 antibody could enhance the antitumor activity of ACT in this model. Cotreatment with both agents increased the number of transferred T cells at the tumor site and also enhanced tumor regressions, compared with treatments with either agent alone. While anti–PD-1 did not reduce the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells present in tumor-bearing mice, we found that it increased expression of IFN-γ and CXCL10 at the tumor site. Bone marrow–transplant experiments using IFN-γR−/− mice implicated IFN-γ as a crucial nexus for controlling PD-1–mediated tumor infiltration by T cells. Taken together, our results imply that blocking the PD-1 pathway can increase IFN-γ at the tumor site, thereby increasing chemokine-dependent trafficking of immune cells into malignant disease sites. Cancer Res; 72(20); 5209–18. ©2012 AACR.

Description: Cancer stem-like cells (CSLC) are crucial in tumor initiation and progression; however, the underlying mechanism for the self-renewal of cancer cells remains undefined. In the study, immunohistochemical analysis of specimens freshly excised from patients with lung adenocarcinoma showed that high expression of insulin-like growth factor I receptor (IGF-IR) in lung adenocarcinoma cells was positively correlated with the expressions of cancer stem cell markers CD133 and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). IGF-IR activation enhanced POU class 5 homeobox 1 (POU5F1) expression on human lung adenocarcinoma stem-like cells (LACSLC) through PI3K/AKT/GSK3β/β-catenin cascade. POU5F1 could form a novel complex with β-catenin and SOX2 to bind Nanog promoter for transcription to maintain self-renewal of LACSLCs, which was dependent on the functional IGF-IR. Genetic and pharmacologic inhibition of IGF-IR abrogated LACSLC capabilities for self-renewal and tumorigenicity in vitro. In an in vivo xenograft tumor model, knockdown of either IGF-IR or POU5F1 impeded tumorigenic potentials of LACSLCs. By analyzing pathologic specimens excised from 200 patients with lung adenocarcinoma, we found that colocalization of highly expressed IGF-IR with β-catenin and POU5F1 predicted poor prognosis. Taken together, we show that IGF-IR—mediated POU5F1 expression to form a complex with β-catenin and SOX2 is crucial for the self-renewal and oncogenic potentials of LACSLCs, and the integrative clinical detection of the expressions of IGF-IR, β-catenin, and POU5F1 is indicatory for predicting prognosis in the patients of lung adenocarcinoma. Cancer Res; 73(10); 3181–9. ©2013 AACR.

Description: Purpose: Giant cell tumors of bone (GCTB) exhibit aggressive bone lytic behavior. Studies have shown that interleukin 17A (IL-17A) is involved pathologic bone resorption in various skeletal disorders. Thus, we have investigated the role of IL-17A in GCTBs. Experimental Design: We evaluated the progression of GCTBs using Campanacci grading and Enneking staging systems in 74 patients with GCTB. The expression of IL-17A and the IL-17A receptor A (IL-17RA) was assessed in GCTB tissues and in both multinucleated giant cells (MNGC) and stromal cells cultured in vitro using immunostaining and reverse transcription PCR (RT-PCR). The effects of IL-17A on the osteolytic activity of the MNGCs and the proliferation of the stromal cells were investigated using the "pit" formation and MTT assays, respectively. The effects of IL-17A on the expression of proosteolytic factors were examined in primary cultured MNGCs and stromal cells using RT-PCR, Western blotting, and gene expression microarrays. Results: In GCTBs, we detected abundant levels of IL-17A, which were associated with tumor extension and grade. IL-17A is predominantly produced by MNGCs, whereas IL-17RA is expressed by both MNGCs and stromal cells in GCTBs. In the MNGCs, the IL-17A increased the mRNA expression of IL-17A and proosteolytic enzymes, and also enhanced osteolytic ability. In the stromal cells, the IL-17A stimulated cellular proliferation and the expression of proosteolytic factors, including RANKL through myc and STAT3, respectively. In addition, IL-17A stimulated in vivo tumor growth and the extent of angiogenesis in GCTBs. Conclusion: IL-17A stimulates the progression of GCTBs and might represent a useful candidate marker for progression and as a therapeutic target for GCTBs. Clin Cancer Res; 19(17); 4697–705. ©2013 AACR .