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Mechanisms underlying the propensity of latent lung adenocarcinoma (LUAD) to relapse are poorly understood. In this study, we show how differential expression of a network of extracellular matrix (ECM) molecules and their interacting proteins contributes to risk of relapse in distinct LUAD subtypes. Overexpression of the hyaluronan receptor HMMR in primary LUAD was associated with an inflammatory molecular signature and poor prognosis. Attenuating HMMR in LUAD cells diminished their ability to initiate lung tumors and distant metastases. HMMR upregulation was not required for dissemination in vivo, but enhanced ECM-mediated signaling, LUAD cell survival, and micrometastasis expansion in hyaluronan-rich microenvironments in the lung and brain metastatic niches. Our findings reveal an important mechanism by which disseminated cancer cells can coopt the inflammatory ECM to persist, leading to brain metastatic outgrowths. Cancer Res; 77(8); 1905–17. ©2017 AACR.

Introduction Increased use of opioids has led to higher rates of overdose and hospital admissions. Studies in trauma populations have focused on outcomes associated with acute intoxications rather than addiction. We hypothesize that clinical outcomes after injury would be inferior for opioid-dependent patients compared to opioid-naive patients. Methods We identified all opioid-dependent adult patients admitted to an academic level I trauma center in 2016 with an Injury Severity Score (ISS) ≥ 5. Patients were further categorized by their pattern of opioid dependency into prescription abuse, illicit abuse, or chronic pain subgroups. Outcome measures included length of stay (LOS), major complications, mortality, non-home discharge, ventilator days, and readmissions. Regression models were adjusted for patient demographics, insurance, ISS, and comorbidities. Results Of the 1450 patients who met the inclusion criteria, 18% were opioid-dependent. Among opioid-dependent patients, 30%, 27%, and 43% were prescription abuse, illicit abuse, and chronic pain patients, respectively. Compared to opioid-naive (non-users) patients, opioid-dependent patients had longer LOS, more ventilator days, more non-home discharges, and higher readmission rates. Subgroup analysis revealed significant differences among all cohorts when compared to non-users in LOS, non-home discharge, readmissions, and major complications. Opioid dependency was not associated with mortality. Conclusion Opioid dependency was detected in 18% of trauma patients and was independently associated with inferior outcomes. The impact of opioid dependency affects each opioid subgroup differently with all cohorts demonstrating increased 30-day readmissions. Opioid dependent patients may be targeted for risk interventions to reduce LOS, non-home discharge, complications and readmissions.

Mechanisms underlying the propensity of latent lung adenocarcinoma (LUAD) to relapse are poorly understood. In this study, we show how differential expression of a network of extracellular matrix (ECM) molecules and their interacting proteins contributes to risk of relapse in distinct LUAD subtypes. Overexpression of the hyaluronan receptor HMMR in primary LUAD was associated with an inflammatory molecular signature and poor prognosis. Attenuating HMMR in LUAD cells diminished their ability to initiate lung tumors and distant metastases. HMMR upregulation was not required for dissemination in vivo, but enhanced ECM-mediated signaling, LUAD cell survival, and micrometastasis expansion in hyaluronan-rich microenvironments in the lung and brain metastatic niches. Our findings reveal an important mechanism by which disseminated cancer cells can coopt the inflammatory ECM to persist, leading to brain metastatic outgrowths. Cancer Res; 77(8); 1905–17. ©2017 AACR.

Background The expansion of Medicaid under the Affordable Care Act extended coverage to any individual with an income up to 138% of the federal poverty level. Our study of surgeon practice management investigated the impact of the type of insurance on access to elective inguinal hernia repair and the disparities in access between Medicaid expansion and nonexpansion states. Methods Practices of 240 hernia repair surgeons across 8 states were randomly selected from the American College of Surgeons Find a Surgeon Database. Investigators posed as simulated patients seeking an evaluation for an inguinal hernia. Physician offices were contacted using a standardized script on separate occasions to assess appointment success rates and waiting periods for 3 different insurance types (BlueCross, Medicaid, Medicare). Results Of 240 surgical practices contacted, 75.4% scheduled appointments for Medicaid patients, compared to 98.8% for Medicare patients and 98.3% for those with private insurance. In states that expanded Medicaid, fewer offices accepted Medicaid patients compared to those in nonexpanded states. No differences in wait times between expanded and nonexpanded states were observed. Surgeons in either solo practices or urban settings were less likely to accept Medicaid patients than those in either group practices or non-urban offices. Conclusions Simulated Medicaid patients were less successful at scheduling appointments for surgical consultation than BlueCross or Medicare patients. Fewer surgical practices in expansion states accepted Medicaid patients despite increased coverage due to Medicaid expansion. These findings should be further investigated amidst future changes in Medicaid to understand their impact on access to surgical care.

SummaryMolecular programs that mediate normal cell differentiation are required for oncogenesis and tumor cell survival in certain cancers. How cell-lineage-restricted genes specifically influence metastasis is poorly defined. In lung cancers, we uncovered a transcriptional program that is preferentially associated with distal airway epithelial differentiation and lung adenocarcinoma (ADC) progression. This program is regulated in part by the lineage transcription factors GATA6 and HOPX. These factors can cooperatively limit the metastatic competence of ADC cells, by modulating overlapping alveolar differentiation and invasogenic target genes. Thus, GATA6 and HOPX are critical nodes in a lineage-selective pathway that directly links effectors of airway epithelial specification to the inhibition of metastasis in the lung ADC subtype.

miR-124 is a brain-enriched microRNA that plays a crucial role in neural development and has been shown to be down-regulated in glioma and medulloblastoma, suggesting its possible involvement in brain tumor progression. Here, we show that miR-124 is down-regulated in a panel of different grades of glioma tissues and in all of the human glioma cell lines we examined. By integrated bioinformatics analysis and experimental confirmation, we identified SNAI2, which is often up-regulated in glioma, as a direct functional target of miR-124. Because SNAI2 has been shown to regulate stem cell functions, we examined the roles of miR-124 and SNAI2 in glioma cell stem-like traits. The results showed that overexpression of miR-124 and knockdown of SNAI2 reduced neurosphere formation, CD133 + cell subpopulation, and stem cell marker (BMI1, Nanog, and Nestin) expression, and these effects could be rescued by re-expression of SNAI2. Furthermore, enhanced miR-124 expression significantly inhibited glioma cell invasion in vitro. Finally, stable overexpression of miR-124 and knockdown of SNAI2 inhibited the tumorigenicity and invasion of glioma cells in vivo. These findings reveal, for the first time, that the tumor suppressor activity of miR-124 could be partly due to its inhibitory effects on glioma stem-like traits and invasiveness through SNAI2. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_subscribed_fulltext

Introduction: Lung cancer is the leading cause of death from cancer in Hong Kong and worldwide. Nearly 80-85% of lung cancers are diagnosed with non-small cell lung cancer (NSCLC). Treatment strategies can be refined according to the distinct difference of NSCLC molecular profiling among different ethnic populations. Moreover, oncogenic driver mutations are closely linked to NSCLC in non-smokers and young people. Therefore, the urge of finding pathogenic mutations is inevitable. This study aims to discover novel mutations in NSCLC patients in Hong Kong by next-generation sequencing and characterize the clinical significance of these mutations. Methods: In order to identify mutations, DNA was purified from 6 pairs of lung tumor-normal tissues from 2 stage I adenocarcinoma (LUAD), 2 stage III LUAD and 2 stage I squamous cell carcinoma (LUSC) patients. Samples were sequenced by targeted sequencing with the TOMA OS-Seq 130 cancer gene panel. Sequence alignment and variant calling were performed by combining TOMA Stratus and in-house pipelines. Results: The mean aligned reads exceeded 14 million and the mean coverage of target region was over 1000X. MUTYH deletion was found in 66.7% (4/6) of the patients. Data from The Cancer Genome Atlas (TCGA) revealed that MUTYH deletion is significantly associated with poorer disease-free survival (DFS) (p=0.0063) and overall survival (p=0.022) in LUAD patients. TCGA data also showed that MUTYH copy number correlates with gene expression (r=0.473) and LUAD patients with MUTYH mRNA overexpression have better DFS (p=0.0205). However, such correlations were not observed in LUSC patients. MUTYH is a DNA glycosylase which involves in the base excision repair and it repairs oxidative DNA damage by preventing G:C to T:A transversion. Numerous studies indicated that biallelic mutations in MUTYH cause MUTYH-associated polyposis and increase the lifetime risk of colorectal cancer. MUTYH mutations could be associated with increased risk of endometrial and breast cancer. Conclusion: To elucidate the clinical significance of MUTYH in LUAD, an independent cohort will be sequenced to validate the primary result. Immunohistochemistry, RT-qPCR and functional assays will be performed to examine the protein expression, the gene expression and the functional role. All these findings suggested that MUTYH deletion may play a crucial role in LUAD and potentially lead to the development of biomarker and therapeutic target. Citation Format: Alvin Hong-Wai Fong, Eunice Yuen-Ting Lau, William Ka-Chun Cheung, William Chi-Shing Cho. Identification of a novel MUTYH mutation in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 560.

Makorin-2 belongs to the makorin RING zinc finger gene family, which encodes putative ribonucleoproteins. Here we cloned the Xenopus makorin-2 (mkrn2) and characterized its function in Xenopus neurogenesis. Forced overexpression of mkrn2 produced tadpoles with dorso-posterior deficiencies and small-head/short-tail phenotype, whereas knockdown of mkrn2 by morpholino antisense oligonucleotides induced double axis in tadpoles. In Xenopus animal cap explant assay, mkrn2 inhibited activin, and retinoic acid induced animal cap neuralization, as evident from the suppression of a pan neural marker, neural cell adhesion molecule. Surprisingly, the anti-neurogenic activity of mkrn2 is independent of the two major neurogenesis signaling cascades, BMP-4 and Wnt8 pathways. Instead, mkrn2 works specifically through the phosphatidylinositol 3-kinase (PI3K) and Akt-mediated neurogenesis pathway. Overexpression of mkrn2 completely abrogated constitutively active PI3K- and Akt-induced, but not dominant negative glycogen synthase kinase-3β (GSK-3β)-induced, neural cell adhesion molecule expression, indicating that mkrn2 acts downstream of PI3K and Akt and upstream of GSK-3β. Moreover, mkrn2 up-regulated the mRNA and protein levels of GSK-3β. These results revealed for the first time the important role of mkrn2 as a new player in PI3K/Akt-mediated neurogenesis during Xenopus embryonic development.

The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo. This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.

Glioblastoma multiforme (GBM), the most severe form of malignant brain tumor, is one of the deadliest cancers identified. Despite multimodal treatments with resection, chemotherapy, and radiotherapy, the prognosis of GBM remains poor over the past 30 years. Thus, the development of new and effective therapies is crucial. In this review, we discuss some alternative strategies for treating GBM by inhibiting the expression of cell cycle-related kinase (CCRK), a protein kinase which has recently been demonstrated to be a candidate oncogene in GBM.

A number of microRNAs (miRNAs) that are evolutionarily conserved not beyond primate lineage have been identified. These primate-specific miRNAs (ps-miRNAs) may attribute to the difference between high-level primates and non-primate mammals or lower vertebrates. Despite of their importance, the genome-wide miRNA conservation patterns and the properties of these ps-miRNAs are largely elusive. In this study, we developed a robust classification system to assess the conservation pattern of all human mature miRNAs across 44 vertebrate genomes. By this comparative genomic analysis, a novel set of 269 ps-miRNAs were identified. We found that many ps-miRNAs were enriched in chromosome 19 and X, forming two main clusters hereafter referred as C19MC and CXMC, respectively. When comparing the seed of ps-miRNAs themselves or with non-ps-miRNAs, more than one half ps-miRNAs sharing common seeds were belonged to C19MC, 9 of which retained a unique seed that had been reported to be enriched in human embryonic stem cells (hESCs) specific miRNAs. Moreover, the most abundant ps-miRNA common seed was possessed by miR-548 family. Most ps-miRNAs had very low expression in adult tissues, which may be attributed to temporal and spatial specific transcript regulation. The ps-miRNAs with relatively high expression were mainly belonged to C19MC and CXMC, and preferentially expressed in hESCs and reproductive system. Sequence anatomy revealed that C19MC ps-miRNAs were highly conserved but not beyond primates and of great sequence similarity. Gene Ontology and KEGG pathway enrichment analyses of predicted target genes indicated that C19MC ps-miRNAs were strongly associated with developmental processes and various cancers. In conclusion, ps-miRNAs may play critical roles in differentiation and growth regulation during early development, especially in maintaining the pluripotency of hESCs. Results from this study may help explaining the differences between primates and lower vertebrates at genetic level.

Makorin-2, consisting of four highly conserved C(3)H zinc fingers, a Cys-His motif and a C(3)HC(4) RING zinc finger domain, is a putative ribonucleoprotein. We have previously reported that Xenopus makorin-2 (mkrn2) is a neurogenesis inhibitor acting upstream of glycogen synthase kinase-3beta (GSK-3beta) in the phosphatidylinositol 3-kinase/Akt pathway. In an effort to identify the functional domains required for its anti-neurogenic activity, we designed and constructed a series of N- and C-terminal truncation mutants of mkrn2. Concurred with the full-length mkrn2, we showed that overexpression of one of the truncation mutants mkrn2(s)-7, which consists of only the third C(3)H zinc finger, Cys-His motif and C(3)HC(4) RING zinc finger, is essential and sufficient to produce the phenotypical dorso-posterior deficiencies and small-head/short-tail phenotype in tadpoles. In animal cap explant assay, we further demonstrated that mkrn2(s)-7 not only inhibits activin and retinoic acid-induced animal cap neuralization and the expression of a pan-neural marker neural cell adhesion molecule, but also induces GSK-3beta expression. These results collectively suggest that the third C(3)H zinc finger, Cys-His motif and C(3)HC(4) RING zinc finger are indispensable for the anti-neurogenic activity of mkrn2.

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133 + side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_OA_fulltext

Nasopharyngeal carcinoma (NPC), a highly metastatic and invasive malignant tumor originating from the nasopharynx, is widely prevalent in Southeast Asia, the Middle East and North Africa. Although viral, dietary and genetic factors have been implicated in NPC, the molecular basis of its pathogenesis is not well defined. Based on a recent microRNA (miRNA) microarray study showing miR-200 downregulation in NPC, we further investigated the role of miR-200a in NPC carcinogenesis. We found that the endogenous miR-200a expression level increases with the degree of differentiation in a panel of NPC cell lines, namely undifferentiated C666-1, high-differentiated CNE-1, and low-differentiated CNE-2 and HNE1 cells. By a series of gain-of-function and loss-of-function studies, we showed that over-expression of miR-200a inhibits C666-1 cell growth, migration and invasion, whereas its knock-down stimulates these processes in CNE-1 cells. In addition, we further identified ZEB2 and CTNNB1 as the functional downstream targets of miR-200a. Interestingly, knock-down of ZEB2 solely impeded NPC cell migration and invasion, whereas CTNNB1 suppression only inhibited NPC cell growth, suggesting that the inhibitory effects of miR-200a on NPC cell growth, migration and invasion are mediated by distinct targets and pathways. Our results reveal the important role of miR-200a as a regulatory factor of NPC carcinogenesis and a potential candidate for miRNA-based therapy against NPC.

Lung cancer is the leading cause of cancer-related deaths worldwide. This poor prognosis is due to the rapid metastatic progression of this disease, yet the mechanisms that govern lung cancer dissemination and colonization are unclear. Through an integrated approach, we identified an alveolar cell-selective gene signature that stratifies lung adenocarcinoma (ADC) into molecular subtypes of distinct prognosis and differentiation states. This transcriptional program is regulated in part by the cell fate transcription factors, GATA6 and HOPX, which are normally required for proper alveolar differentiation. In an experimental model, suppression of GATA6 and HOPX cooperatively enhances metastatic competence of lung ADC cells. Whole genome RNA sequencing reveals that this transcriptional network can modulate specific target genes and pathways involved in airway epithelial differentiation and invasion. Our findings identify a novel cell lineage molecular program whose perturbation enhances lung cancer metastasis. Citation Format: William K.C. Cheung, Minghui Zhao, Zongzhi Liu, Paul D. Cao, Laura E. Stevens, Thomas F. Westbrook, Don X. Nguyen. A lineage-selective transcriptional pathway linking lung differentiation and cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3872. doi:10.1158/1538-7445.AM2013-3872

Lung cancer is the leading cause of cancer-related deaths worldwide. This poor prognosis is due to the rapid metastatic progression of this disease, yet the mechanisms that govern lung cancer dissemination and colonization are unclear. Through an integrated approach, we identified an alveolar cell-selective gene signature that stratifies lung adenocarcinoma (ADC) into molecular subtypes of distinct prognosis and differentiation states. This transcriptional program is regulated in part by the cell fate transcription factors, GATA6 and HOPX, which are normally required for proper alveolar differentiation. In an experimental model, suppression of GATA6 and HOPX cooperatively enhances metastatic competence of lung ADC cells. Whole genome RNA sequencing reveals that this transcriptional network can modulate specific target genes and pathways involved in airway epithelial differentiation and invasion. Our findings identify a novel cell lineage molecular program whose perturbation enhances lung cancer metastasis. Citation Format: William K.C. Cheung, Minghui Zhao, Zongzhi Liu, Paul D. Cao, Laura E. Stevens, Thomas F. Westbrook, Don X. Nguyen. A lineage-selective transcriptional pathway linking lung differentiation and cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B56.