Serine-Threonine Kinase Receptor Associate Protein (STRAP) confers an aggressive phenotype in neuroblastoma via regulation of Focal Adhesion Kinase (FAK).

BACKGROUND: Serine-threonine kinase receptor associated protein (STRAP), a scaffolding protein, is upregulated in many solid tumors. As such, we hypothesized that STRAP may be overexpressed in neuroblastoma tumors and may play a role in neuroblastoma tumor progression. METHODS: We examined two publicly available neuroblastoma patient databases, GSE49710 (n = 498) and GSE49711 (n = 498), to investigate STRAP expression in human specimens. SK-N-AS and SK-N-BE(2) human neuroblastoma cell lines were stably transfected with STRAP overexpression (OE) plasmid, and their resulting phenotype studied. PamChip® kinomic peptide microarray evaluated the effects of STRAP overexpression on kinase activation. RESULTS: In human specimens, higher STRAP expression correlated with high-risk disease, unfavorable histology, and decreased overall neuroblastoma patient survival. STRAP OE in neuroblastoma cell lines led to increased proliferation, growth, supported a stem-like phenotype and activated downstream FAK targets. When FAK was targeted with the small molecule FAK inhibitor, PF-573,228, STRAP OE neuroblastoma cells had significantly decreased growth compared to control empty vector cells. CONCLUSION: Increased STRAP expression in neuroblastoma was associated with unfavorable tumor characteristics. STRAP OE resulted in increased kinomic activity of FAK. These findings suggest that the poorer outcomes in neuroblastoma tumors associated with STRAP overexpression may be secondary to FAK activation.

Non-coding small nucleolar RNA SNORD17 promotes the progression of hepatocellular carcinoma through a positive feedback loop upon p53 inactivation.

Recent evidence suggests that small nucleolar RNAs (snoRNAs) are involved in the progression of various cancers, but their precise roles in hepatocellular carcinoma (HCC) remain largely unclear. Here, we report that SNORD17 promotes the progression of HCC through a positive feedback loop with p53. HCC-related microarray datasets from the Gene Expression Omnibus (GEO) database and clinical HCC samples were used to identify clinically relevant snoRNAs in HCC. SNORD17 was found upregulated in HCC tissues compared with normal liver tissues, and the higher expression of SNORD17 predicted poor outcomes in patients with HCC, especially in those with wild-type p53. SNORD17 promoted the growth and tumorigenicity of HCC cells in vitro and in vivo by inhibiting p53-mediated cell cycle arrest and apoptosis. Mechanistically, SNORD17 anchored nucleophosmin 1 (NPM1) and MYB binding protein 1a (MYBBP1A) in the nucleolus by binding them simultaneously. Loss of SNORD17 promoted the translocation of NPM1 and MYBBP1A into the nucleoplasm, leading to NPM1/MDM2-mediated stability and MYBBP1A/p300-mediated activation of p53. Interestingly, p300-mediated acetylation of p53 inhibited SNORD17 expression by binding to the promoter of SNORD17 in turn, forming a positive feedback loop between SNORD17 and p53. Administration of SNORD17 antisense oligonucleotides (ASOs) significantly suppressed the growth of xenograft tumors in mice. In summary, this study suggests that SNORD17 drives cancer progression by constitutively inhibiting p53 signaling in HCC and may represent a potential therapeutic target for HCC.

Serine-threonine Kinase Receptor-Associated Protein is a Critical Mediator of APC Mutation-Induced Intestinal Tumorigenesis Through a Feed-Forward Mechanism.

BACKGROUND & AIMS: Inactivation of the Apc gene is a critical early event in the development of sporadic colorectal cancer (CRC). Expression of serine-threonine kinase receptor-associated protein (STRAP) is elevated in CRCs and is associated with poor outcomes. We investigated the role of STRAP in Apc mutation-induced intestinal tumor initiation and progression. METHODS: We generated Strap intestinal epithelial knockout mice (StrapΔIEC) by crossing mice containing floxed alleles of Strap (Strapfl/fl) with Villin-Cre mice. Then we generated ApcMin/+;Strapfl/fl;Vill-Cre (ApcMin/+;StrapΔIEC) mice for RNA-sequencing analyses to determine the mechanism of function of STRAP. We used human colon cancer cell lines (DLD1, SW480, and HT29) and human and mouse colon tumor-derived organoids for STRAP knockdown and knockout and overexpression experiments. RESULTS: Strap deficiency extended the average survival of ApcMin/+ mice by 80 days and decreased the formation of intestinal adenomas. Expression profiling revealed that the intestinal stem cell signature, the Wnt/ß-catenin signaling, and the MEK/ERK pathway are down-regulated in Strap-deficient adenomas and intestinal organoids. Correlation studies suggest that these STRAP-associated oncogenic signatures are conserved across murine and human colon cancer. STRAP associates with MEK1/2, promotes binding between MEK1/2 and ERK1/2, and subsequently induces the phosphorylation of ERK1/2. STRAP activated Wnt/ß-catenin signaling through MEK/ERK-induced phosphorylation of LRP6. STRAP was identified as a target of mutated Apc and Wnt/ß-catenin signaling as chromatin immunoprecipitation and luciferase assays revealed putative binding sites of the ß-catenin/TCF4 complex on the Strap promoter. CONCLUSIONS: STRAP is a target of, and is required in, Apc mutation/deletion-induced intestinal tumorigenesis through a novel feed-forward STRAP/MEK-ERK/Wnt-ß-catenin/STRAP regulatory axis.

Serine-Threonine Kinase Receptor-Associated Protein (STRAP) Knockout Decreases the Malignant Phenotype in Neuroblastoma Cell Lines.

Background: Serine-threonine kinase receptor-associated protein (STRAP) plays an important role in neural development but also in tumor growth. Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid malignancy of childhood and it continues to carry a poor prognosis. The recent discovery of the role of STRAP in another pediatric solid tumor, osteosarcoma, and the known function of STRAP in neural development, led us to investigate the role of STRAP in neuroblastoma tumorigenesis. Methods: STRAP protein expression was abrogated in two human neuroblastoma cell lines, SK-N-AS and SK-N-BE(2), using transient knockdown with siRNA, stable knockdown with shRNA lentiviral transfection, and CRISPR-Cas9 genetic knockout. STRAP knockdown and knockout cells were examined for phenotypic alterations in vitro and tumor growth in vivo. Results: Cell proliferation, motility, and growth were significantly decreased in STRAP knockout compared to wild-type cells. Indicators of stemness, including mRNA abundance of common stem cell markers Oct4, Nanog, and Nestin, the percentage of cells expressing CD133 on their surface, and the ability to form tumorspheres were significantly decreased in the STRAP KO cells. In vivo, STRAP knockout cells formed tumors less readily than wild-type tumor cells. Conclusion: These novel findings demonstrated that STRAP plays a role in tumorigenesis and maintenance of neuroblastoma stemness.

A novel approach for monitoring TGF-ß signaling in vivo in colon cancer.

The TGF-ß receptor kinase inhibitors (TRKI) have been reported to inhibit tumorigenicity in colon cancer. However, there is no direct evidence showing that these inhibitors function through inhibiting the TGF-ß- mediated tumor-promoting effects in vivo. We established a TGF-ß inducible reporter system by inserting a luciferase reporter gene to the vector downstream of TGF-ß-inducible promoter elements, and transfected it into colon cancer cell lines. TRKIs SB431542 and LY2109761 were used to treat TGF-ß inducible cells in vitro and in vivo. The luciferase activity was induced 5.24-fold by TGF-ß in CT26 inducible cells, while it was marginally changed in MC38 inducible cells lacking Smad4 expression. Temporary treatment of mice with SB431542 inhibited the TGF-ß pathway and TGF-ß induced bioluminescence activity in vivo. Long-term treatment with LY2109761 inhibited tumorigenicity and liver metastasis in vivo in concomitant with reduced luciferase activity in the tumor. In this study, we established a model to monitor the TGF-ß pathway in vivo and to compare the antitumor effects of TRKIs. Based on this novel experimental tool, we provided direct evidences that LY2109761 inhibits tumorigenicity and liver metastasis by blocking the pro-oncogenic functions of TGF-ß in vivo.

STRAP regulates alternative splicing fidelity during lineage commitment of mouse embryonic stem cells.

Alternative splicing (AS) is involved in cell fate decisions and embryonic development. However, regulation of these processes is poorly understood. Here, we have identified the serine threonine kinase receptor-associated protein (STRAP) as a putative spliceosome-associated factor. Upon Strap deletion, there are numerous AS events observed in mouse embryoid bodies (EBs) undergoing a neuroectoderm-like state. Global mapping of STRAP-RNA binding in mouse embryos by enhanced-CLIP sequencing (eCLIP-seq) reveals that STRAP preferably targets transcripts for nervous system development and regulates AS through preferred binding positions, as demonstrated for two neuronal-specific genes, Nnat and Mark3. We have found that STRAP involves in the assembly of 17S U2 snRNP proteins. Moreover, in Xenopus, loss of Strap leads to impeded lineage differentiation in embryos, delayed neural tube closure, and altered exon skipping. Collectively, our findings reveal a previously unknown function of STRAP in mediating the splicing networks of lineage commitment, alteration of which may be involved in early embryonic lethality in mice.

TRIP13 promotes metastasis of colorectal cancer regardless of p53 and microsatellite instability status.

Overexpression of TRIP13, a member of the AAA-ATPase family, is linked with various cancers, but its role in metastasis is unknown in colorectal cancer (CRC). In the current study, we investigated the role TRIP13 in experimental metastasis and its involvement in regulation of WNT/ß-catenin and EGFR signaling pathways. Evaluation of formalin-fixed paraffin-embedded (FFPE) and frozen tissues of adenomas and CRCs, along with their corresponding normal samples, showed that TRIP13 was gradually increased in its phenotypic expression from adenoma to carcinoma and that its overexpression in CRCs was independent of patient's gender, age, race/ethnicity, pathologic stage, and p53 and microsatellite instability (MSI) status. Moreover, liver metastases of CRCs showed TRIP13 overexpression as compared to matched adjacent liver tissues, indicating the biological relevance of TRIP13 in CRC progression and metastasis. TRIP13 knockdown impeded colony formation, invasion, motility, and spheroid-forming capacity of CRC cells irrespective of their p53 and MSI status. Furthermore, xenograft studies demonstrated high expression of TRIP13 contributed to tumor growth and metastasis. Depletion of TRIP13 in CRC cells decreased metastasis and it was independent of the p53 and MSI status. Furthermore, TRIP13 interacted with a tyrosine kinase, FGFR4; this interaction could be essential for activation of the EGFR-AKT pathway. In addition, we demonstrated the involvement of TRIP13 in the Wnt signaling pathway and in the epithelial-mesenchymal transition. Cell-based assays revealed that miR-192 and PNPT1 regulate TRIP13 expression in CRC. Additionally, RNA sequencing of CRC cells with TRIP13 knockdown identified COL6A3, TREM2, SHC3, and KLK7 as downstream targets that may have functional relevance in TRIP13-mediated tumor growth and metastasis. In summary, our results demonstrated that TRIP13 promotes tumor growth and metastasis regardless of p53 and MSI status, and indicated that it is a target for therapy of CRC.

MiR-216b/Smad3/BCL-2 Axis Is Involved in Smoking-Mediated Drug Resistance in Non-Small Cell Lung Cancer.

Epidemiologic studies have shown that vast majority of lung cancers (85-90%) are causally linked to tobacco smoking. Although much information has been gained about the effects of smoking on various signaling pathways, little is known about how deregulation of miRNAs leads to activation of oncogenes and inhibition of tumor suppressor genes in non-small cell lung cancer (NSCLC). Our previous study showed that smoking inhibits TGF-ß-induced tumor suppressor functions through downregulation of Smad3 in lung cancer cells. In order to understand the upstream mechanism of downregulation of Smad3 by smoking, we performed miRNA microarray analyses after treating human lung adenocarcinoma A549 and immortalized peripheral lung epithelial HPL1A cells with cigarette smoke condensate (CSC). We identified miR-216b as being upregulated in CSC treated cells. MiR-216b overexpression decreases Smad3 protein expression by binding to its 3'-UTR, and attenuates transforming growth factor beta (TGF-ß) signaling and target gene expression. MiR-216b increases B-cell lymphoma 2 (BCL-2) expression and promotes chemoresistance of NSCLC cells by decreasing apoptosis. Increased acetylation of histones H3 and H4 in miR-216b gene promoter plays a role in CSC induced miR-216b expression. Taken together, these results suggest that smoking-mediated upregulation of miR-216b increases NSCLC cell growth by downregulating Smad3 and inhibiting TGF-ß-induced tumor suppressor function, and induces resistance to platinum-based therapy.

PTPRε Acts as a Metastatic Promoter in Hepatocellular Carcinoma by Facilitating Recruitment of SMAD3 to TGF-ß Receptor 1.

BACKGROUND AND AIMS: Transforming growth factor beta (TGF-ß) suppresses early stages of tumorigenesis, but contributes to the migration and metastasis of cancer cells. However, the role of TGF-ß signaling in invasive prometastatic hepatocellular carcinoma (HCC) is poorly understood. In this study, we investigated the roles of canonical TGF-ß/mothers against decapentaplegic homolog 3 (SMAD3) signaling and identified downstream effectors on HCC migration and metastasis. APPROACH AND RESULTS: By using in vitro trans-well migration and invasion assays and in vivo metastasis models, we demonstrated that SMAD3 and protein tyrosine phosphatase receptor epsilon (PTPRε) promote migration, invasion, and metastasis of HCC cells in vitro and in vivo. Further mechanistic studies revealed that, following TGF-ß stimulation, SMAD3 binds directly to PTPRε promoters to activate its expression. PTPRε interacts with TGFBR1/SMAD3 and facilitates recruitment of SMAD3 to TGFBR1, resulting in a sustained SMAD3 activation status. The tyrosine phosphatase activity of PTPRε is important for binding with TGFBR1, recruitment and activation of SMAD3, and its prometastatic role in vitro. A positive correlation between pSMAD3/SMAD3 and PTPRε expression was determined in HCC samples, and high expression of SMAD3 or PTPRε was associated with poor prognosis of patients with HCC. CONCLUSIONS: PTPRε positive feedback regulates TGF-ß/SMAD3 signaling to promote HCC metastasis.

Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.

Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.

Molecular characterization of pro-metastatic functions of ß4-integrin in colorectal cancer.

The ß4-integrin subunit has been implicated in development and progression of several epithelial tumor types. However, its role in metastases of colorectal cancer (CRC) remains elusive. To study CRC metastasis, we generated a highly invasive, metastatic cell line MC38-LM10 (LM10) by passaging mouse CRC MC38 cells ten times, using a splenic injection model of liver metastasis. Affymetrix microarray analyses of LM10 and MC38 cell lines and their corresponding liver metastases generated a gene signature for CRC metastasis. This signature shows strong upregulation of ß4-integrin in LM10 cells and corresponding metastases. Upregulation of ß4-integrin in highly aggressive LM10 cells is associated with increased migration, invasion, and liver metastases. Furthermore, stable knockdown of ß4-integrin in human CRC SW620 cells reduces Bcl-2 expression, increases apoptosis, and decreases invasion, tumorigenicity, and liver metastasis, thus resulting in significantly increased survival of mice (hazard ratio = 0.32, 95% confidence interval = 0.15-0.66, P<0.01). Patients with CRC tumors display higher ß4-integrin levels in stages 1-4 and significantly lower survival rate. Collectively, ß4-integrin plays a critical role in CRC progression, invasion, and metastasis, suggesting that it could be a potential therapeutic target for CRC patients.

Regulation of EMT in Colorectal Cancer: A Culprit in Metastasis.

Epithelial to mesenchymal transition (EMT) is a process during which cells lose their epithelial characteristics, for instance cell polarity and cell-cell contact, and gain mesenchymal properties, such as increased motility. In colorectal cancer (CRC), EMT is associated with an invasive or metastatic phenotype. In this review, we discuss recent studies exploring novel regulation mechanisms of EMT in CRC, including the identification of new CRC EMT regulators. Upregulation of inducers can promote EMT, leading to increased invasiveness and metastasis in CRC. These inducers can downregulate E-cadherin and upregulate N-cadherin and vimentin (VIM) through modulating EMT-related signaling pathways, for instance WNT/ß-catenin and TGF-ß, and EMT transcription factors, such as zinc finger E-box binding homeobox 1 (ZEB1) and ZEB2. In addition, several microRNAs (miRNAs), including members of the miR-34 and miR-200 families, are found to target mRNAs of EMT-transcription factors, for example ZEB1, ZEB2, or SNAIL. Downregulation of these miRNAs is associated with distant metastasis and advanced stage tumors. Furthermore, the role of EMT in circulating tumor cells (CTCs) is also discussed. Mesenchymal markers on the surface of EMT CTCs were found to be associated with metastasis and could serve as potential biomarkers for metastasis. Altogether, these studies indicate that EMT is orchestrated by a complicated network, involving regulators of different signaling pathways. Further studies are required to understand the mechanisms underlying EMT in CRC.

STRAP Promotes Stemness of Human Colorectal Cancer via Epigenetic Regulation of the NOTCH Pathway.

NOTCH signaling exerts essential roles in normal and malignant intestinal physiology and the homeostasis of cancer stem-like cells (CSC), but the basis for this latter role remains obscure. The signaling scaffold protein STRAP is upregulated in several cancers, where it promotes tumorigenicity and metastasis. Here we report a novel oncogenic function for STRAP in maintaining CSC subpopulations in a heterogeneous mixture by antagonizing formation of the chromatin modifier PRC2 and by epigenetically activating NOTCH signals in human colorectal cancer. Silencing STRAP sensitized colorectal cancer cells to chemotherapeutic drugs in vitro and in vivo STRAP depletion also contributed to a reduced stem-like phenotype of colorectal cancer cells, as indicated by reduced expression of the CSC signature and NOTCH signaling regulators in vitro and by diminished tumorigenesis in vivo Genes encoding some upstream activators of NOTCH were highly enriched for H3K27me3, which forms repressive chromatin domains upon STRAP silencing. Mechanistically, STRAP competitively disrupted association of the PRC2 subunits EZH2 and SUZ12, thereby inhibiting PRC2 assembly. Restoring the NOTCH pathway by lentiviral expression of NICD1 or HES1 in STRAP-depleted tumor cells reversed the CSC phenotype. In 90 colorectal cancer clinical specimens, a significant positive correlation was documented between the expression of STRAP and HES1. Overall, our findings illuminated a novel STRAP-NOTCH1-HES1 molecular axis as a CSC regulator in colorectal cancer, with potential implications to improve treatment of this disease. Cancer Res; 77(20); 5464-78. ©2017 AACR.

Role of TGF-ß-induced Claudin-4 expression through c-Jun signaling in non-small cell lung cancer.

Claudin-4 has been identified as an integral member of tight junctions and has been found to be upregulated in various types of cancers especially in metastatic cancers. However, the molecular mechanism of the upregulation of Claudin-4 and its role in lung tumorigenesis are unknown. The aim of the present study is to investigate the role of Claudin-4 on migration and tumorigenicity of lung cancer cells and to examine the regulatory effects of TGF-ß on Claudin-4 expression. We have observed that TGF-ß induces the expression of Claudin-4 dramatically in lung cell lines in a time dependent manner. TGF-ß-induced Smad signaling is important for enhancing Claudin-4 mRNA level through inducing its promoter activity. Treatment with curcumin, a c-Jun inhibitor, or stable knockdown of c-Jun abrogates TGF-ß-induced Claudin-4 expression suggesting an involvement of the c-Jun pathway. Notably, TGF-ß-induced Claudin-4 expression through c-Jun pathway plays a role in TGF-ß-mediated motility and tumorigenicity of these cells. In support of these observations, we have uncovered that Claudin-4 is upregulated in 14 of 24 (58%) lung tumors when compared with normal lung tissue. This is the first study to show how TGF-ß regulates the expression of Claudin-4 through c-Jun signaling and how this pathway contributes to the migratory and tumorigenic phenotype of lung tumor cells.

Effect of Cigarette Smoking on Epithelial to Mesenchymal Transition (EMT) in Lung Cancer.

Epithelial to mesenchymal transition (EMT) is a process that allows an epithelial cell to acquire a mesenchymal phenotype through multiple biochemical changes resulting in an increased migratory capacity. During cancer progression, EMT is found to be associated with an invasive or metastatic phenotype. In this review, we focus on the discussion of recent studies about the regulation of EMT by cigarette smoking. Various groups of active compounds found in cigarette smoke such as polycyclic aromatic hydrocarbons (PAH), nicotine-derived nitrosamine ketone (NNK), and reactive oxygen specicies (ROS) can induce EMT through different signaling pathways. The links between EMT and biological responses to cigarette smoke, such as hypoxia, inflammation, and oxidative damages, are also discussed. The effect of cigarette smoke on EMT is not only limited to cancer types directly related to smoking, such as lung cancer, but has also been found in other types of cancer. Altogether, this review emphasizes the importance of understanding molecular mechanisms of the induction of EMT by cigarette smoking and will help in identifying novel small molecules for targeting EMT induced by smoking.

Activin A-Smad Signaling Mediates Connective Tissue Growth Factor Synthesis in Liver Progenitor Cells.

Liver progenitor cells (LPCs) are activated in chronic liver damage and may contribute to liver fibrosis. Our previous investigation reported that LPCs produced connective tissue growth factor (CTGF/CCN2), an inducer of liver fibrosis, yet the regulatory mechanism of the production of CTGF/CCN2 in LPCs remains elusive. In this study, we report that Activin A is an inducer of CTGF/CCN2 in LPCs. Here we show that expression of both Activin A and CTGF/CCN2 were upregulated in the cirrhotic liver, and the expression of Activin A positively correlates with that of CTGF/CCN2 in liver tissues. We go on to show that Activin A induced de novo synthesis of CTGF/CCN2 in LPC cell lines LE/6 and WB-F344. Furthermore, Activin A contributed to autonomous production of CTGF/CCN2 in liver progenitor cells (LPCs) via activation of the Smad signaling pathway. Smad2, 3 and 4 were all required for this induction. Collectively, these results provide evidence for the fibrotic role of LPCs in the liver and suggest that the Activin A-Smad-CTGF/CCN2 signaling in LPCs may be a therapeutic target of liver fibrosis.

Novel role of STRAP in progression and metastasis of colorectal cancer through Wnt/ß-catenin signaling.

Serine-Threonine Kinase Receptor-Associated Protein (STRAP) interacts with a variety of proteins and influences a wide range of cellular processes. Aberrant activation of Wnt/ß-catenin signaling has been implicated in the development of colorectal cancer (CRC). Here, we show the molecular mechanism by which STRAP induces CRC metastasis by promoting ß-catenin signaling through its stabilization. We have genetically engineered a series of murine and human CRC and lung cancer cell lines to investigate the effects of STRAP on cell migration and invasion in vitro, and on tumorigenicity and metastasis in vivo. Downregulation of STRAP inhibits invasion, tumorigenicity, and metastasis of CRC cells. Mechanistically, STRAP binds with GSK-3ß and reduces the phosphorylation, ubiquitylation, and degradation of ß-catenin through preventing its binding to the destruction complex. This leads to an inhibition of Wnt/ß-catenin signaling and reduction in the expression of downstream targets, such as Cyclin D1, matrix metalloproteinases 2 and 9, and ß-TrCP. In human CRC specimens, higher STRAP expression correlates significantly with ß-catenin expression with increased nuclear levels (R =0.696, p < .0001, n =128). Together, these results suggest that STRAP increases invasion and metastasis of CRC partly through inhibiting ubiquitin-dependent degradation of ß-catenin and promoting Wnt/ß-catenin signaling.

An epigenetic auto-feedback loop regulates TGF-ß type II receptor expression and function in NSCLC.

The downregulation of transforming growth factor-ß (TGF-ß) type II receptor (TßRII) expression and function plays a pivotal role in the loss of the TGF-ß-induced tumor suppressor function that contributes to lung cancer progression. The aberrant expression of miRNAs has been shown to be involved in the regulation of oncogenes and tumor suppressor genes. Our current study involving miRNA microarray, northern blot and QRT-PCR analysis shows an inverse correlation between miR-20a and TßRII expression in non-small cell lung cancer (NSCLC) tissues and cell lines. Stable expression of miR-20a downregulates TßRII in lung epithelial cells which results in an inhibition of TGF-ß signaling and attenuation of TGF-ß-induced cell growth suppression and apoptosis. Stable knock down of miR-20a increases TßRII expression and inhibits tumorigenicity of lung cancer cells in vivo. Oncogene c-Myc promotes miR-20a expression by activating its promoter leading to downregulation of TßRII expression and TGF-ß signaling. MiR-145, which is upregulated by TGF-ß, inhibits miR-20a expression by targeting c-Myc and upregulates TßRII expression. These correlations among miRNAs and cellular proteins are supported by TCGA public database using NSCLC specimens. These results suggest a novel mechanism for the loss of TßRII expression and TGF-ß-induced tumor suppressor functions in lung cancer through a complex auto-feedback loop TGF-ß/miR-145/c-Myc/miR-20a/TßRII.

Oncogenic STRAP functions as a novel negative regulator of E-cadherin and p21(Cip1) by modulating the transcription factor Sp1.

We have previously reported the identification of a novel WD-domain protein, STRAP that plays a role in maintenance of mesenchymal morphology by regulating E-cadherin and that enhances tumorigenicity partly by downregulating CDK inhibitor p21(Cip1). However, the functional mechanism of regulation of E-cadherin and p21(Cip1) by STRAP is unknown. Here, we have employed STRAP knock out and knockdown cell models (mouse embryonic fibroblast, human cancer cell lines) to show how STRAP downregulates E-cadherin and p21(Cip1) by abrogating the binding of Sp1 to its consensus binding sites. Moreover, ChIP assays suggest that STRAP recruits HDAC1 to Sp1 binding sites in p21(Cip1) promoter. Interestingly, loss of STRAP can stabilize Sp1 by repressing its ubiquitination in G1 phase, resulting in an enhanced expression of p21(Cip1) by >4.5-fold and cell cycle arrest. Using Bioinformatics and Microarray analyses, we have observed that 87% mouse genes downregulated by STRAP have conserved Sp1 binding sites. In NSCLC, the expression levels of STRAP inversely correlated with that of Sp1 (60%). These results suggest a novel mechanism of regulation of E-cadherin and p21(Cip1) by STRAP by modulating Sp1-dependent transcription, and higher expression of STRAP in lung cancer may contribute to downregulation of E-cadherin and p21(Cip1) and to tumor progression.

Reduced expression of transcriptional intermediary factor 1 gamma promotes metastasis and indicates poor prognosis of hepatocellular carcinoma.

UNLABELLED: Transcriptional intermediary factor 1 gamma (TIF1γ) may play either a potential tumor-suppressor or -promoter role in cancer. Here we report on a critical role of TIF1γ in the progression of hepatocellular carcinoma (HCC). Reduced expression of TIF1γ was detected in HCC, especially in advanced HCC tissues, compared to adjacent noncancerous tissues. HCC patients with low TIF1γ expression had shorter overall survival times and higher recurrence rates than those with high TIF1γ expression. Reduced TIF1γ expression was an independent and significant risk factor for recurrence and survival after curative resection. In HCC cells, TIF1γ played a dual role: It promoted tumor growth in early-stage HCC, but not in advanced-stage HCC, whereas it inhibited invasion and metastasis in both early- and advanced-stage HCC. Mechanistically, we confirmed that TIF1γ inhibited transforming growth factor-ß/ Drosophila mothers against decapentaplegic protein (TGF-ß/Smad) signaling through monoubiquitination of Smad4 and suppressed the formation of Smad2/3/4 complex in HCC cells. TGF-ß-inducing cytostasis and metastasis were both inhibited by TIF1γ in HCC. We further proved that TIF1γ suppressed cyotstasis-related TGF-ß/Smad downstream c-myc down-regulation, as well as p21/cip1 and p15/ink4b up-regulation in early-stage HCC. Meanwhile, TGF-ß inducible epithelial-mesenchymal transition and TGF-ß/Smad downstream metastatic cascades, including phosphatase and tensin homolog deleted on chromosome ten down-regulation, chemokine (CXC motif) receptor 4 and matrix metalloproteinase 1 induction, and epidermal growth factor receptor- and protein kinase B-signaling transactivation, were inhibited by TIF1γ. In addition, we found that the down-regulation of TIF1γ in HCC was caused by hypermethylation of CpG islands in the TIF1γ promoter, and demonstrated that the combination of TIF1γ and phosphorylated Smad2 was a more powerful predictor of poor prognosis. CONCLUSION: TIF1γ regulates tumor growth and metastasis through inhibition of TGF-ß/Smad signaling and may serve as a novel prognostic biomarker in HCC.