Purpose: Leptomeningeal metastasis (LM) is a detrimental complication of non-small cell lung cancer (NSCLC) and associated with poor prognosis. However, the underlying mechanisms of the metastasis process are still poorly understood. Experimental Design: We performed next-generation panel sequencing of primary tumor tissue, cerebrospinal fluid (CSF), and matched normal controls from epidermal growth factor receptor (EGFR) mutation-positive NSCLC patients with LM. Results: The status of EGFR-activating mutations was highly concordant between primary tumor and CSF. PIK3CA aberrations were high in these patients, implicating an association with LM risk. Intriguingly, low overlapping of somatic protein-changing variants was observed between paired CSF and primary lesions, exhibiting tumor heterogeneity and genetic divergence. Moreover, genes with CSF-recurrent genomic alterations were predominantly involved in cell-cycle regulation and DNA-damage response (DDR), suggesting a role of the pathway in LM development. Conclusions: Our study has shed light on the genomic variations of NSCLC-LM, demonstrated genetic heterogeneity and divergence, uncovered involvement of cell-cycle and DDR pathway, and paved the way for potential therapeutic approaches to this unmet medical need. Clin Cancer Res; 24(1); 209-16. (C) 2017 AACR.
Purpose: Leukemia stem cells (LSCs) are an important source of tyrosine kinase inhibitor resistance and disease relapse in patients with chronic myelogenous leukemia (CML). Targeting LSCs may be an attractive strategy to override this thorny problem. Given that EZH2 was overexpressed in primary CML CD34 thorn cells, our purpose in this study was to evaluate the effects of targeting EZH2 on CML LSCs and clarify its underlying mechanism. Experimental Design: Human primary CML CD34 thorn cells and retrovirally BCR-ABL-driven CML mouse models were employed to evaluate the effects of suppression of EZH2 by GSK126-or EZH2-specific shRNA in vitro and in vivo. Recruitment of EZH2 and H3K27me3 on the promoter of tumorsuppressor gene PTEN in CML cells wasmeasured by chromatin immunoprecipitation assay. Results: Our results showed that pharmacologic inhibition of EZH2 by GSK126 not only elicited apoptosis and restricted cell growth in CML bulk leukemia cells, but also decreased LSCs in CML CD34 thorn cells while sparing those from normal bone marrow CD34 thorn cells. Suppression of EZH2 by GSK126 or specific shRNA prolonged survival of CML mice and reduced the number of LSCs in mice. EZH2 knockdown resulted in elevation of PTEN and led to impaired recruitment of EZH2 and H3K27me3 on the promoter of PTEN gene. The effect of EZH2 knockdown in the CML mice was at least partially reversed by PTEN knockdown. Conclusions: These findings improve the understanding of the epigenetic regulation of stemness in CML LSCs and warrant clinical trial of GSK126 in refractory patients with CML. Clin Cancer (C) 2017 AACR.
Purpose: Agents extracted from natural sources with antitumor property have attracted considerable attention from researchers and clinicians because of their safety, efficacy, and immediate availability. Degalactotigonin (DGT), extracted from Solanum nigrum L., has anticancer properties without serious side effects. Here, we explored whether DGT can inhibit the growth and metastasis of osteosarcoma. Experimental Design: MTT, colony formation, and apoptosis assays were performed to analyze the effects of DGT on osteosarcoma cell viability in vitro. The migration and invasion abilities were measured using a Transwell assay. Animal models were used to assess the roles of DGT in both tumor growth and metastasis of osteosarcoma. Gli1 expression and function were measured in osteosarcoma cells and clinical samples. After DGT treatment, Gli1 activation and the phosphorylation status of multiple cellular kinases were measured with a luciferase reporter and phospho-kinase antibody array. Results: DGT inhibited proliferation, induced apoptosis, and suppressed migration and invasion in osteosarcoma cells. DGT, injected intraperitoneally after tumor inoculation, significantly decreased the volume of osteosarcoma xenografts and dramatically diminished the occurrence of osteosarcoma xenograft metastasis to the lungs. Mechanistically, DGT inhibited osteosarcoma growth and metastasis through repression of the Hedgehog/Gli1 pathway, which maintains malignant phenotypes and is involved in the prognosis of osteosarcoma patients. DGT decreased the activity of multiple intracellular kinases that affect the survival of osteosarcoma patients, including GSK3b. In addition, DGT represses the Hedgehog/Gli1 pathway mainly through GSK3b inactivation. Conclusions: Our studies provide evidence that DGT can suppress the growth and metastasis of human osteosarcoma through modulation of GSK3b inactivation-mediated repression of the Hedgehog/Gli1 pathway. (C) 2017 AACR.
Purpose: The response rate of patients with head and neck squamous cell carcinoma (HNSCC) to cetuximab therapy is only 15% to 20%, despite frequent EGFR overexpression. Because immunosuppression is common in HNSCC, we hypothesized that adding a proinflammatory TLR8 agonist to cetuximab therapy might result in enhanced T-lymphocyte stimulation and anti-EGFR-specific priming. Experimental Design: Fourteen patients with previously untreated HNSCC were enrolled in this neoadjuvant trial and treated preoperatively with 3 to 4 weekly doses of motolimod (2.5 mg/m(2)) and cetuximab. Correlative tumor and peripheral blood specimens were obtained at baseline and at the time of surgical resection and analyzed for immune biomarker changes. Preclinical in vitro studies were also performed to assess the effect of cetuximab plus motolimod on myeloid cells. Results: TLR8 stimulation skewed monocytes toward an M1 phenotype and reversed myeloid-derived suppressor cell (MDSC) suppression of T-cell proliferation in vitro. These data were validated in a prospective phase Ib neoadjuvant trial, in which fewer MDSC and increased M1 monocyte infiltration were found in tumor-infiltrating lymphocytes. Motolimod plus cetuximab also decreased induction of Treg and reduced markers of suppression, including CTLA-4, CD73, and membrane-bound TGFb. Significantly increased circulating EGFR-specific T cells were observed, concomitant with enhanced CD8 thorn T-cell infiltration into tumors. These T cells manifested increased T-cell receptor (TCR) clonality, upregulation of the costimulatory receptor CD27, and downregulation of inhibitory receptor TIGIT. Conclusions: Enhanced inflammatory stimulation in the tumor microenvironment using a TLR agonist overcomes suppressive myeloid and regulatory cells, enhancing the cellular antitumor immune response by therapeutic mAb in HNSCC. (C) 2017 AACR.
Purpose: Metabolic reprogramming is frequently identified in hepatocellular carcinoma (HCC), which is the most common type of liver malignancy. The reprogrammed cellular metabolisms promote tumor cell survival, proliferation, angiogenesis, and metastasis. However, the mechanisms of this process remain unclear in HCC. Experimental Design: The global nontargeted metabolic study in 69 paired hepatic carcinomas and adjacent tissue specimens was performed using capillary electrophoresis-time of flight mass spectrometry-based approach. Key findings were validated by targeted metabolomic approach. Biological studies were also performed to investigate the role of proline biosynthesis in HCC pathogenesis. Results: Proline metabolism was markedly changed in HCC tumor tissue, characterized with accelerated consumption of proline and accumulation of hydroxyproline, which significantly correlated with a-fetoprotein levels and poor prognosis in HCC. In addition, we found that hydroxyproline promoted hypoxia- and HIF-dependent phenotype in HCC. Moreover, we demonstrated that hypoxia activated proline biosynthesis via upregulation of ALDH18A1, subsequently leading to accumulation of hydroxyproline via attenuated PRODH2 activity. More importantly, we showed that glutamine, proline, and hydroxyproline metabolic axis supported HCC cell survival through modulating HIF1 alpha stability in response to hypoxia. Finally, inhibition of proline biosynthesis significantly enhanced cytotoxicity of sorafenib in vitro and in vivo. Conclusions: Our results demonstrate that hypoxic microenvironment activates proline metabolism, resulting in accumulation of hydroxyproline that promotes HCC tumor progression and sorafenib resistance through modulating HIF1 alpha. These findings provide the proof of concept for targeting proline metabolism as a potential therapeutic strategy for HCC. (C) 2017 AACR.
Purpose: Regulated in development and DNA damage response-1 (REDD1) is a stress-related protein and is involved in the progression of cancer. The role and regulatory mechanism of REDD1 in bladder urothelial carcinoma (BUC), however, is yet unidentified. Experimental Design: The expression of REDD1 in BUC was detected by Western blot analysis and immunohistochemistry (IHC). The correlation between REDD1 expression and clinical features in patients with BUC were assessed. The effects of REDD1 on cellular proliferation, apoptosis, autophagy, and paclitaxel sensitivity were determined both in vitro and in vivo. Then the targeted-regulating mechanism of REDD1 by miRNAs was explored. Results: Here the significant increase of REDD1 expression is detected in BUC tissue, and REDD1 is first reported as an independent prognostic factor in patients with BUC. Silencing REDD1 expression in T24 and EJ cells decreased cell proliferation, increased apoptosis, and decreased autophagy, whereas the ectopic expression of REDD1 in RT4 and BIU87 cells had the opposite effect. In addition, the REDD1-mediated proliferation, apoptosis, and autophagy are found to be negatively regulated by miR-22 in vitro, which intensify the paclitaxel sensitivity via inhibition of the well-acknowledged REDD1-EEF2K autophagy axis. AKT/mTOR signaling initially activated or inhibited in response to silencing or enhancing REDD1 expression and then recovered rapidly. Finally, the inhibited REDD1 expression by either RNAi or miR-22 sensitizes BUC tumor cells to paclitaxel in a subcutaneous transplant carcinoma model in vivo. Conclusions: REDD1 is confirmed as an oncogene in BUC, and antagonizing REDD1 could be a potential therapeutic strategy to sensitize BUC cells to paclitaxel. (C) 2017 AACR.
Purpose: The success of immune checkpoint inhibitors strengthens the notion that tumor growth and regression are immune regulated. To determine whether distinct tissue immune microenvironments differentially affect clinical outcome in non-small cell lung cancer (NSCLC), an extended analysis of PD-L1 and tumor-infiltrating lymphocytes (TIL) was performed. Experimental Design: Samples from resected adenocarcinoma (ADC 42), squamous cell carcinoma (SCC 58), and 26 advanced diseases (13 ADC and 13 SCC) treated with nivolumab were analyzed. PD-L1 expression and the incidence of CD3, CD8, CD4, PD-1, CD57, FOXP3, CD25, and Granzyme B TILs were immunohistochemically assessed. Results: PD-L1 levels inversely correlated with N involvement, although they did not show a statistically significant prognostic value in resected patients. The incidence and phenotype of TILs differed in SCC versus ADC, in which EGFR and KRAS mutations conditioned a different frequency and tissue localization of lymphocytes. NSCLC resected patients with high CD8(pos) lymphocytes lacking PD-1 inhibitory receptor had a longer overall survival (OS:HR = 2.268; 95% CI, 1.056-4.871, P = 0.03). PD-1-to-CD8 ratio resulted in a prognostic factor both on univariate (HR = 1.952; 95% CI, 1.34-3.12, P = 0.001) and multivariate(HR = 1.943; 95% CI, 1.38-2.86, P = 0.009) analysis. Moreover, low PD-1 incidence among CD8(pos) cells was a distinctive feature of nivolumab-treated patients, showing clinical benefit with a prolonged progression-free survival (PFS: HR = 4.51; 95% CI, 1.45-13.94, P = 0.004). Conclusions: In the presence of intrinsic variability in PD-L1 expression, the reservoir of PD-1-negative effector T lymphocytes provides an immune-privileged microenvironment with a positive impact on survival of patients with resected disease and response to immunotherapy in advanced NSCLC. (C) 2017 AACR.
Purpose: To explore the molecular mechanism and prognosis of bone-invasive pituitary adenomas (BIPA). Experimental design: A total of 274 patients with pituitary adenomas were followed up. Transcriptomic microarrays analysis was performed on 10 pituitary adenomas, including five BIPAs and five non-bone-invasive pituitary adenomas (NBIPA). The targeted molecular markers were validated by qRT-PCR, IHC, ELISA, and osteoclast differentiation. Results: Clinical variable analyses revealed a significant correlation between bone invasion and female sex, large tumor volume, non-gross total resection (NGTR), and tumor regrowth. BIPAs had worse progression-free survival (PFS) than did NBIPAs in the NGTR and nonfunctional pituitary adenoma (NFPA) groups. Gene ontology functional and KEGG pathway analyses showed that the biological processes and pathways were primarily immune and inflammatory pathways. Pathway act work showed that osteoclast differentiation pathway was significantly implicated in the pathway network. BIPAs had higher expression of TNF alpha than that of NBIPAs on IHC. In vitro, TNF alpha could induce RAW264.7 cells to differentiate into mature osteoclasts, leading to bone destruction. NR_033258, lncRNA SNHG24, miR-181c-5p, and miR-454-3p can regulate TNF alpha expression. Conclusions: BIPAs had worse PFS than did NBIPAs in the NGTR and NFPA groups. Inflammatory and immune factors play an important role in BIPAs. TNF alpha can directly induce osteoclast differentiation in BIPAs. NR_033258, lncRNA SNHG24, miR-181c-5p, and miR-454-3p can regulate TNF alpha expression. TNF alpha and its related lncRNAs and miRNAs represent potential therapeutic targets for bone-invasive pituitary adenomas in the future. (C) 2018 AACR.
Purpose: Inflammatory infiltration plays important roles in both carcinogenesis and metastasis. We are interested in understanding the inhibitory mechanism of metformin on tumor-associated inflammation in prostate cancer. Experimental Design: By using a transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model, in vitro macrophage migration assays, and patient samples, we examined the effect of metformin on tumor-associated inflammation during the initiation and after androgen deprivation therapy of prostate cancer. Results: Treating TRAMP mice with metformin delays prostate cancer progression from low-grade prostatic intraepithelial neoplasia to high-grade PIN, undifferentiated to well-differentiated, and PIN to adenocarcinoma with concurrent inhibition of inflammatory infiltration evidenced by reduced recruitment of macrophages. Furthermore, metformin is capable of inhibiting the following processes: inflammatory infiltration after androgen deprivation therapy (ADT) induced by surgically castration in mice, bicalutamide treatment in patients, and hormone deprivation in LNCaP cells. Mechanistically, metformin represses inflammatory infiltration by downregulating both COX2 and PGE2 in tumor cells. Conclusions: Metformin is capable of repressing prostate cancer progression by inhibiting infiltration of tumor-associated macrophages, especially those induced by ADT, by inhibiting the COX2/PGE2 axis, suggesting that a combination of ADT with metformin could be a more efficient therapeutic strategy for prostate cancer treatment. (C) 2018 AACR.
Purpose: Current tumor-node-metastasis (TNM) staging system cannot provide adequate information for prediction of prognosis and chemotherapeutic benefits. We constructed a classifier to predict prognosis and identify a subset of patients who can benefit from adjuvant chemotherapy. Experimental Design: We detected expression of 15 immunohistochemistry (IHC) features in tumors from 251 gastric cancer (GC) patients and evaluated the association of their expression level with overall survival (OS) and disease-free survival (DFS). Then, integrating multiple clinicopathologic features and IHC features, we used support vector machine (SVM)-based methods to develop a prognostic classifier (GC-SVM classifier) with features. Further validation of theGC-SVM classifier was performed in two validation cohorts of 535 patients. Results: The GC-SVM classifier integrated patient sex, carcinoembryonic antigen, lymph node metastasis, and the protein expression level of eight features, including CD3(invasive margin) (IM), CD3(center of tumor (CT)), CD8(IM), CD45RO(CT), CD57(IM), CD66b(IM), CD68(CT), and CD34. Significant differences were found between the high-and low-GC-SVM patients in 5-year OS and DFS in training and validation cohorts. Multivariate analysis revealed that the GC-SVM classifier was an independent prognostic factor. The classifier had higher predictive accuracy for OS and DFS than TNM stage and can complement the prognostic value of the TNM staging system. Further analysis revealed that stage II and III GC patients with high-GC-SVM were likely to benefit from adjuvant chemotherapy. Conclusions: The newly developed GC-SVM classifier was a powerful predictor of OS and DFS. Moreover, the GC-SVM classifier could predict which patients with stage II and III GC benefit from adjuvant chemotherapy. (C) 2018 AACR.
Purpose: Long noncoding RNAs have been implicated in gliomagenesis, but theirmechanisms of action are mainly undocumented. Through public glioma mRNA expression data sets, we found that NEAT1 was a potential oncogene. We systematically analyzed the clinical significance and mechanism of NEAT1 in glioblastoma. Experimental Design: Initially, we evaluated whether NEAT1 expression levels could be regulated by EGFR pathway activity. We subsequently evaluated the effect of NEAT1 on the WNT/beta-catenin pathway and its target binding gene. The animal model supported the experimental findings. Results: We found that NEAT1 levels were regulated by EGFR pathway activity, which was mediated by STAT3 and NF kappa B (p65) downstream of the EGFR pathway. Moreover, we found that NEAT1 was critical for glioma cell growth and invasion by increasing beta-catenin nuclear transport and down-regulating ICAT, GSK3B, and Axin2. Taken together, we found that NEAT1 could bind to EZH2 and mediate the trimethylation of H3K27 in their promoters. NEAT1 depletion also inhibited GBM cell growth and invasion in the intracranial animal model. Conclusions: The EGFR/NEAT1/EZH2/beta-catenin axis serves as a critical effector of tumorigenesis and progression, suggesting new therapeutic directions in glioblastoma. (C) 2017 AACR.
Purpose: The spatial heterogeneity of phenotypic and molecular characteristics of CTCs within the circulatory system remains unclear. Herein, we mapped the distribution and characterized biological features of CTCs along the transportation route in hepatocellular carcinoma (HCC). Experimental Design: In 73 localizedHCCpatients, blood was drawn from peripheral vein (PV), peripheral artery (PA), hepatic veins (HV), infrahepatic inferior vena cava (IHIVC), and portal vein (PoV) before tumor resection. Epithelial and mesenchymal transition (EMT) phenotype in CTCs were analyzed by a 4-channel immunofluorescence CellSearch assay and microfluidic quantitative RT-PCR. The clinical significance of CTCs from different vascular sites was evaluated. Results: The CTC number and size gradient between tumor efferent vessels and postpulmonary peripheral vessels was marked. Tracking the fate of CTC clusters revealed that CTCs displayed an aggregated-singular-aggregated manner of spreading. Single-cell characterization demonstrated that EMT status of CTCs was heterogeneous across different vascular compartments. CTCs were predominantly epithelial at release, but switched to EMT-activated phenotype during hematogeneous transit via Smad2 and beta-catenin related signaling pathways. EMT activation in primary tumor correlated with total CTC number at HV, rather than epithelial or EMT-activated subsets of CTCs. Follow-up analysis suggested that CTC and circulating tumor microemboli burden in hepatic veins and peripheral circulation prognosticated postoperative lung metastasis and intrahepatic recurrence, respectively. Conclusions: The current data suggested that a profound spatial heterogeneity in cellular distribution and biological features existed among CTCs during circulation. Multivascular measurement of CTCs could help to reveal novel mechanisms of metastasis and facilitate prediction of postoperative relapse or metastasis pattern in HCC. (C) 2017 AACR.
Purpose: Epithelial and endothelial tyrosine kinase (Etk), also known as bone marrow X kinase (Bmx), was found to be critical in modulating the chemoresistance of small-cell lung cancer (SCLC) in our preliminary study. However, the molecular mechanisms of Etk in SCLC chemoresistance remain poorly understood. Experimental Design: We determined correlation of Etk with autophagy in SCLC. And direct inhibition of autophagy was performed to validate its effect on chemoresistance. Coimmuno-precipitation (co-IP) and GST-pull down experiments were conducted to verify the interaction of Etk and PFKFB4, after a microarray analysis. In vitro and in vivo gain or loss-of-function analyses and evaluation of PFKFB4 expression in SCLC specimens, were done to validate its role in chemoresistance. Ibrutinib was administrated in SCLC cells to verify its synergistic anti-tumor effect with chemotherapy using preclinical models including a PDX model. Results: Downregulation of Etk suppressed autophagy in chemoresistant SCLC cells, and direct inhibition of autophagy sensitized cells to chemotherapy. PFKFB4 (6-phosphofructo-2kinase/fructose-2,6-biphosphatase 4) was identified as a downstream target of Etk and an Etk-interacting protein, which promoted chemoresistance in SCLC and was associated with poor therapeutic response and prognosis. Furthermore, ibrutinib was found to exhibit a synergistic anti-tumor effect with chemotherapy in targeting Etk. Conclusions: Our results demonstrated for the first time that Etk interacts with PFKFB4 to promote SCLC chemoresistance through regulation of autophagy. Aberrant Etk and PFKFB4 can be predictive factors for the chemotherapy response as well as potential therapeutic targets in SCLC. (C) 2017 AACR.
Purpose: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. In China, chronic hepatitis B virus (HBV) infection remains the major risk factor for HCC. In this study, we performed a genome-wide association study (GWAS) among Chinese populations to identify novel genetic loci contributing to susceptibility to HBV-related HCC. Experimental Design: GWAS scan is performed in a collection of 205 HBV-related HCC trios (each trio includes an affected proband and his/her both parents), and 355 chronic HBV carriers with HCC (cases) and 360 chronic HBV carriers without HCC (controls), followed by two rounds of replication studies totally consisting of 3,796 cases and 2,544 controls. Results: We identified a novel association signal within the CDK14 gene at 7q21.13 (index rs10272859, OR = 1.28, P = 9.46 x 10(-10)). Furthermore, we observed that the at-risk rs10272859[G] allele was significantly associated with higher mRNA expression levels of CDK14 in liver tissues. Chromosome conformation capture assays in liver cells confirmed that a physical interaction exists between the promoter region of CDK14 and the risk-associated SNPs in strong linkage disequilibrium with the index rs10272859 at 7q21.13. This index rs10272859 also showed significant association with the survival of HCC patients. Conclusions: Our findings highlight a novel locus at 7q21.13 conferring both susceptibility and prognosis to HBV-related HCC, and suggest the CDK14 gene to be the functional target of the 7q21.13 locus. (C) 2017 AACR.