Tumor Suppressor Candidate 1 Suppresses Cell Growth and Predicts Better Survival in Glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor with poor prognosis and limited treatment options. Tumor suppressor candidate 1 (TUSC1) was recently identified as a potential tumor suppressor in human cancers. However, the expression and potential function of TUSC1 in GBM remain unclear. Herein, we report that TUSC1 is significantly decreased in GBM tissues and cell lines. Patients with high levels of TUSC1 displayed a significant better survival compared with those with low levels of TUSC1. Functional experiments demonstrated that exogenous expression of TUSC1 inhibited GBM cell proliferation and induced G1 phase arrest by down-regulating CDK4. Moreover, overexpression of TUSC1 retarded tumor growth in vivo. Together, our findings revealed that TUSC1 might be a crucial tumor suppressor gene and a novel therapeutic target for GBM.     

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.     

The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.     

Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Decreased TUSC3 Promotes Pancreatic Cancer Proliferation,Invasion and Metastasis

Pancreatic cancer is an aggressive disease with dismal prognosis. It is of paramount importance to understand the underlying etiological mechanisms and identify novel, consistent, and easy-to-apply prognostic factors for precision therapy. TUSC3 (tumor suppressor candidate 3) was identified as a potential tumor suppressor gene and previous study showed TUSC3 is decreased in pancreatic cancer at mRNA level, but its putative tumor suppressor function remains to be verified. In this study, TUSC3 expression was found to be suppressed both at mRNA and protein levels in cell line models as well as in clinical samples; decreased TUSC3 expression was associated with higher pathological TNM staging and poorer outcome. In three pairs of cell lines with different NF-κB activity, TUSC3 expression was found to be reversely correlated with NF-κB activity. TUSC3-silenced pancreatic cancer cell line exhibited enhanced potential of proliferation, migration and invasion. In an orthotopic implanted mice model, TUSC3 silenced cells exhibited more aggressive phenotype with more liver metastasis. In conclusion, the current study shows that decreased immunological TUSC3 staining is a factor prognostic of poor survival in pancreatic cancer patients and decreased TUSC3 promotes pancreatic cancer cell proliferation, invasion and metastasis. The reverse correlation between NF-κB activity and TUSC3 expression may suggest a novel regulation pattern for this molecule.     

Human Breast Cancer Cell Lines Co-Express Neuronal,Epithelial, and Melanocytic Differentiation Markers In Vitro and In Vivo

Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3) and basal (MDA-MB-231) types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL) and glioblastoma cell lines (U87 and D54). These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.     

Cyr61, a member of CCN family, is a tumor suppressor in non-small cell lung cancer

Cysteine-rich protein 61 (Cyr61) is a member of a family of growth factor-inducible immediate-early genes. It regulates cell adhesion, migration, proliferation, and differentiation and is involved in tumor growth. In our experiments, the role of Cyr61 in non-small cell lung cancer (NSCLC) was examined. Expression of Cyr61 mRNA was decreased markedly in four of five human lung tumor samples compared with their normal matched lung samples. NSCLC cell lines NCI-H520 and H460, which have no endogenous Cyr61, formed 60-90% fewer colonies after being transfected with a Cyr61 cDNA expression vector than cells transfected with the same amount of empty vector. After stable transfection of a Cyr61 cDNA expression vector, proliferation of both H520-Cyr61 and H460-Cyr61 sublines decreased remarkably compared with the cells stably transfected with empty vector. The addition of antibody against Cyr61 partially rescued the growth suppression of both H520-Cyr61 and H460-Cyr61 cells. Cell cycle analysis revealed that both H520-Cyr61 and H460-Cyr61 cells developed G(1) arrest, prominently up-regulated expression of p53 and p21(WAF1), and had decreased activity of cyclin-dependent kinase 2. The increase of pocket protein pRB2/p130 was also detected in these cells. Notably, both of the Cyr61-stably transfected lung cancer cell lines developed smaller tumors than those formed by the wild-type cells in nude mice. Taken together, we conclude that Cyr61 may play a role as a tumor suppressor in NSCLC.     

Knockdown of Lymphoid Enhancer factor 1 Inhibits Colon Cancer Progression In Vitro and In Vivo

Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.     

Array-Based Approaches for the Identification of Epigenetic Silenced Tumor Suppressor Genes

Carcinogenesis involves the inactivation or inhibition of genes that function as tumor suppressors. Deletions, mutations, or epigenetic silencing of tumor suppressor genes can lead to altered growth, differentiation, and apoptosis. DNA methylation and histone modifications are important epigenetic mechanisms of gene regulation and play essential roles both independently and cooperatively in tumor initiation and progression. Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated discovery of novel tumor suppressor genes. One of the most useful of these approaches is an epigenetic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase (HDAC) inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Recently, using cancer cell lines treated with a DNA methylation inhibitor and/or a HDAC inhibitor in conjunction with cDNA microarray analysis, candidate tumor suppressor genes, which are subject to epigenetic silencing, have been identified in endometrial, colorectal, esophageal, and pancreatic cancers. An increasing number of studies have utilized epigenetic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

Overexpression of N-Myc Downstream-Regulated Gene 2 (NDRG2) Regulates the Proliferation and Invasion of Bladder Cancer Cells In Vitro and In Vivo

N-Myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor gene, which plays an important role in controlling tumor growth. The aim of this study was to investigate the expression of NDRG2 gene in bladder cancer (BC) tissues and several bladder cancer cell lines, and to seek its clinical and pathological significance. Ninety-seven bladder carcinoma and 15 normal bladder tissue sections were analyzed retrospectively with immunohistochemistry. The human bladder cancer cell line T24 was infected with LEN-NDRG2 or LEN-LacZ. The effects of NDRG2 overexpression on T24 cells and T24 nude mouse xenografts were measured via cell growth curves, tumor growth curves, flow cytometric analysis, western blot and Transwell assay. NDRG2 was highly expressed in normal bladder tissue, but absent or rarely expressed in cacinomatous tissues (χ²=8.761, p < 0.01). The NDRG2 level was negatively correlated with tumor grade and pathologic stage(r=-0.248, p < 0.05), as well as increased c-myc level (r=-0.454, p< 0.001). The expression of NDRG2 was low in the three BC cell lines. T24 cells infected with LEN-NDRG2 showed inhibition of proliferation both in vitro and in vivo, and NDRG2 overexpression can inhibit tumor growth and invasion in vitro.     

Astrocytes Directly Influence Tumor Cell Invasion and Metastasis In Vivo

Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2) and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.     

Functional Polymorphism of the CK2α Intronless Gene Plays Oncogenic Roles in Lung Cancer

Protein kinase CK2 is frequently up-regulated in human cancers, although the mechanism of CK2 activation in cancer remains unknown. In this study, we investigated the role of the CK2α intronless gene (CSNK2A1P, a presumed CK2α pseudogene) in the pathogenesis of human cancers. We found evidence of amplification and over-expression of the CSNK2A1P gene in non- small cell lung cancer and leukemia cell lines and 25% of the lung cancer tissues studied. The mRNA expression levels correlated with the copy numbers of the CSNK2A1P gene. We also identified a novel polymorphic variant (398T/C, I133T) of the CSNK2A1P gene and showed that the 398T allele is selectively amplified over the 398C allele in 101 non-small cell lung cancer tissue samples compared to those in 48 normal controls (p = 0.013<0.05). We show for the first time CSNK2A1P protein expression in transfected human embryonic kidney 293T and mouse embryonic fibroblast NIH-3T3 cell lines. Both alleles are transforming in these cell lines, and the 398T allele appears to be more transforming than the 398C allele. Moreover, the 398T allele degrades PML tumor suppressor protein more efficiently than the 398C allele and shows a relatively stronger binding to PML. Knockdown of the CSNK2A1P gene expression with specific siRNA increased the PML protein level in lung cancer cells. We report, for the first time, that the CSNK2A1P gene is a functional proto-oncogene in human cancers and its functional polymorphism appears to degrade PML differentially in cancer cells. These results are consistent with an important role for the 398T allele of the CSNK2A1P in human lung cancer susceptibility.     

Protein Secretion Is Required for Pregnancy-Associated Plasma Protein-A to Promote Lung Cancer Growth In Vivo

Pregnancy-associated plasma protein-A (PAPPA) has been reported to regulate the activity of insulin-like growth factor (IGF) signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs) thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE) tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression.     

Adenoviral Delivery of the EMX2 Gene Suppresses Growth in Human Gastric Cancer

Background

EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration.

Results

In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector.

Conclusion

Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer.     

Gremlin is Overexpressed in Lung Adenocarcinoma and Increases Cell Growth and Proliferation in Normal Lung Cells

Background

Gremlin, a member of the Dan family of BMP antagonists, is a glycosylated extracellular protein. Previously Gremlin has been shown to play a role in dorsal-ventral patterning, in tissue remodeling, and recently in angiogenesis. Evidence has previously been presented showing both over- and under-expression of Gremlin in different tumor tissues. Here, we sought to quantify expression of Gremlin in cancers of the lung and performed in vitro experiments to check whether Gremlin promotes cell growth and proliferation.

Methodology/Principal Findings

Expression of Gremlin in 161 matched tumor and normal lung cancer specimens is quantified by quantitative real-time PCR and protein level is measured by immunohistochemistry. GREM1 was transfected into lung fibroblast and epithelial cell lines to assess the impact of overexpression of Gremlin in vitro.

Results

Lung adenocarcinoma but not squamous cell carcinoma shows a significant increase in Gremlin expression by mRNA and protein level. Lung fibroblast and epithelial cell lines transfected with GREM1 show significantly increased cell proliferation.

Conclusions/Significance

Our data suggest that Gremlin acts in an oncogenic manner in lung adenocarcinoma and could hold promise as a new diagnostic marker or potential therapeutic target in lung AD or general thoracic malignancies.     

Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans

Background

Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.

Methods

Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.

Results

Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).

Conclusions

DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00059605","term_id":"NCT00059605"}}NCT00059605