Potential Tumor Suppressor Role Research Articles

Synergistic repression of thyroid hyperplasia by cyclin C and Pten.

The cyclin C-Cdk8 kinase has been identified as both a tumor suppressor and an oncogene depending on the cell type. The genomic locus encoding cyclin C (Ccnc) is often deleted in aggressive anaplastic thyroid tumors. To test for a potential tumor suppressor role for cyclin C, Ccnc alone, or Ccnc in combination with a previously described thyroid tumor suppressor Pten, was deleted late in thyroid development. Although mice harboring individual Pten or Ccnc deletions exhibited modest thyroid hyperplasia, the double mutant demonstrated dramatic thyroid expansion resulting in animal death by 22 weeks. Further analysis revealed that Ccncthyr-/- tissues exhibited a reduction in signal transducer and activator of transcription 3 (Stat3) phosphorylation at Ser727. Further analysis uncovered a post-transcriptional requirement of both Pten and cyclin C in maintaining the levels of the p21 and p53 tumor suppressors (also known as CDKN1A and TP53, respectively) in thyroid tissue. In conclusion, these data reveal the first tumor suppressor role for cyclin C in a solid tumor model. In addition, this study uncovers new synergistic activities of Pten and cyclin C to promote quiescence through maintenance of p21 and p53.

The Pattern of Malignancies in Down Syndrome and Its Potential Context With the Immune System.

The immune surveillance theory of cancer posits that the body's immune system detects and destroys randomly occurring malignant cells. This theory is based on the observation of the increased frequency of malignancies in primary and secondary immunodeficiencies, and is supported by the successful demonstration of immune augmentation in current oncological immune therapy approaches. We review this model in the context of Down syndrome (DS), a condition with a unique tumor profile and various immune defects. Children and adults with DS are more prone to infections due to anatomical reasons and a varying degree of T- and B-cell maturation defects, NK cell dysfunction, and chemotactic or phagocytic abnormalities. However, despite an increased incidence of lymphoblastic and myeloblastic leukemia of infants and children with DS, individuals with DS have a globally decreased incidence of solid tumors as compared to age-adjusted non-DS controls. Additionally, cancers that have been considered “proof of immune therapy principles,” such as renal carcinoma, small cell lung carcinoma, and malignant melanoma, are less frequent in adults with DS compared to the general population. Thus, despite the combination of an increased risk of leukemia with detectable immune biological abnormalities and a clinical immunodeficiency, people with DS appear to be protected against many cancers. This observation does not support the immune surveillance theory in the context of DS and indicates a potential tumor-suppressive role for trisomy 21 in non-hematological malignancies.

FBXL3 is regulated by miRNA-4735-3p and suppresses cell proliferation and migration in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common type of primary lung cancer and regarded as cancer killer. The aim of this study was to discover the detailed function and molecular mechanism of F-box and leucine rich repeat protein 3 (FBXL3) in NSCLC. In this study, the expression level of FBXL3 in NSCLC tissues and cell lines was firstly examined and identified. Moreover, the relationship between FBXL3 and the overall survival rate of NSCLC patients was analyzed by Kaplan–Meier survival curve. Functionally, MTT, colony formation assay and transwell assays were performed to determine the role of FBXL3 in regulating NSCLC cell proliferation, migration and invasion. The proliferation and migration were suppressed by overexpression of FBXL3, indicating the potential tumor suppressive role of FBXL3 in NSCLC. In addition, the dual-luciferase reporter and RNA pull-down assays revealed that miR-4735-3p was a novel upstream modulator of FBXL3. Further study showed that miR-4735-3p was upregulated in NSCLC tissues and cell lines. Finally, rescue assays and function assays revealed that miR-4735-3p exerted oncogenic function in NSCLC, and this function can be attenuated by FBXL3. Taken together, FBXL3 was regulated by miR-4735-3p and suppressed cell proliferation and invasion in non-small cell lung cancer.

Activation of PAR4 Upregulates p16 through Inhibition of DNMT1 and HDAC2 Expression via MAPK Signals in Esophageal Squamous Cell Carcinoma Cells.

A previous study showed that a downexpression of protease-activated receptor 4 (PAR4) is associated with the development of esophageal squamous cell carcinoma (ESCC). In this study, we explored the relationship between PAR4 activation and the expression of p16, and elucidated the underlying mechanisms in PAR4 inducing the tumor suppressor role in ESCC. ESCC cell lines (EC109 and TE-1) were treated with PAR4-activating peptide (PAR4-AP). Immunohistochemistry for DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) was performed in 26 cases of ESCC tissues. We found that DNMT1 and HDAC2 immunoreactivities in ESCC were significantly higher than those in adjacent noncancerous tissues. PAR4 activation could suppress DNMT1 and HDAC2, as well as increase p16 expressions, whereas silencing PAR4 dramatically increased HDAC2 and DNMT1, as well as reduced p16 expressions. Importantly, the chromatin immunoprecipitation-PCR (ChIP-PCR) data indicated that treatment of ESCC cells with PAR4-AP remarkably suppressed DNMT1 and HDAC2 enrichments on the p16 promoter. Furthermore, we demonstrated that activation of PAR4 resulted in an increase of p38/ERK phosphorylation and activators for p38/ERK enhanced the effect of PAR4 activation on HDAC2, DNMT1, and p16 expressions, whereas p38/ERK inhibitors reversed these effects. Moreover, we found that activation of PAR4 in ESCC cells significantly inhibited cell proliferation and induced apoptosis. These findings suggest that PAR4 plays a potential tumor suppressor role in ESCC cells and represents a potential therapeutic target of this disease.

Shuttling SLC2A4RG is Regulated by 14-3-3θ¸ to Modulate Cell Survival Via Caspase-3 and Caspase-6 in Human Glioma

Glioma is the most common and aggressive primary brain tumor with polygenic susceptibility. The cytoplasmic/nuclear shuttling protein, SLC2A4RG (SLC2A4 regulator), has been identified in the 20q13.33 region influencing glioma susceptibility by genome-wide association studies (GWAS) and fine mapping analyses. In this study, we revealed that nuclear SLC2A4RG was significantly decreased in human glioma specimens and associated with patients' unfavorable prognosis. The potential tumor suppressor role of SLC2A4RG was further validated by in vitro and in vivo experiments that SLC2A4RG could attenuate cell proliferation via G2/M phase arrest and induce glioma cell apoptosis by direct transactivation of caspase-3 and caspase-6. Moreover, its function displaying showed to depend on the nuclear transportation of SLC2A4RG, however, bound with 14-3-3θ¸, it would be sequestered in the cytoplasm followed by reversal effect. We, therefore, identify a new pro-oncogenic mechanism whereby 14-3-3I¸ negatively regulates the nuclear function of the tumor suppressor SLC2A4RG, with significant therapeutic implications for the intervention of human glioma. Funding: This work was supported by the National Natural Science Foundation of China (81372706, 81572501, 81071739, 81372235, and 81201978). Declaration of Interest: No potential conflicts of interest were disclosed. Ethical Approval: The present study was approved by the Ethical Review Committee of SMMU.

Wnt Effector TCF4 Is Dispensable for Wnt Signaling in Human Cancer Cells.

T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

Abstract 2426: Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is a neuroendocrine malignancy, caused by activating mutations of the RET proto-oncogene, with frequent lymph node and distant metastasis at diagnosis. Tyrosine kinase inhibitors (TKIs) that target the RET have proven effective for treatment of MTC, but resistance to TKIs evolves in half of the treated patients. We have previously shown that the expression levels of Activating Transcription Factor 4 (ATF4) which is a stress-induced transcription factor, significantly decreased or lost in half of the MTC tumors. We also demonstrated that forced expression of ATF4 or TKIs-induced level of ATF4 decreases survival of MTC cells by promoting degradation of RET, blocking the activation of RET downstream signaling pathways and subsequently induces apoptosis. More importantly, ATF4 knockdown by shRNA decreased the sensitivity to tyrosine kinase inhibitor-induced apoptosis. Further thyroid gland of Atf4-knockout mice showed C-cell hyperplasia, a precancerous lesion for MTC suggesting a potential tumor suppressor role of ATF4 in MTC. Stress-induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a feature of specialized secretory cells, including MTC cells that secrete calcitonin. ATF4 promotes the induction of apoptosis under persistent stress conditions, although the mechanism is not clearly understood. We hypothesize that the combination of ATF4 inducer and TKIs causes an excessive cellular oxidative stress resulting in an activation of apoptosis, all of which may prevent resistance to TKIs. Here, we show that the ER-associated protein degradation (ERAD) inhibitor, eeyarestatin sensitizes MTC cells to the TKIs including sunitinib and vandetanib. The combination of eeyarestatin and TKIs causes a synergistic induction of ATF4 expression and its target genes, an accumulation of reactive oxygen species and subsequent cell death. Chromatin immunoprecipitation followed by sequencing assay (ChIP-seq) in MTC cells treated with eeyarestatin and immunoprecipitated with ATF4 and acetylated lysine 9 of histone 3, identified transcription Kruppel-like factor 9 (KLF9) gene as a transcriptional target of ATF4 activation. KLF9 plays a functional role in oxidative stress-induced cell death. Treatment with eeyarestatin and vandetanib alone or in combination increases the occupancy of ATF4 at the promoter of KLF9 gene and stimulates the expression of KLF9. Depletion of ATF4 by shRNA leads to downregulation of KLF9 expression and prevents oxidative stress-induced cell death. These findings suggest that induction of the ATF4/KLF9 axis causes oxidative stress leading to excessive ER stress, and subsequent cell death. Thus, combining TKIs and ERAD inhibitors that promote ATF4 levels could be effective therapeutic strategies for treating MTC and preventing resistance to TKIs. Citation Format: Rozita Bagheri-Yarmand, Krishna M. Sinha, Ling Li, Yue Lu, Robert F. Gagel. Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid 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 2426.

Risk stratification of gastrointestinal stromal tumors by CtBP2 and CD44 analysis.

48 Background: C-terminal binding protein 2 (CtBP2) is an oncogenic transcription factor that promotes cancer stem cell (CSC) growth and self-renewal, and controls pathways for tumor initiation, progression, and response to therapy. Expression of CtBP2 is linked to aggressive behavior in ovarian cancer, while genetic or pharmacologic targeting of CtBP2 disrupts CSC growth and self-renewal. CD44 is a cell surface marker that has been linked to CSC populations in many solid tumors, though data is conflicting on its role as tumor suppressor or oncogene. Gastrointestinal stromal tumors (GIST) is an increasingly common malignancy where risk stratification is essential for guiding post-surgical adjuvant therapy. We hypothesized that CtBP2 and CD44 staining would enhance risk stratification of GIST which currently relies on purely non-molecular clinicopathologic criteria. Methods: We identified 149 GIST cases from 1990 to 2016 in the Virginia Commonwealth University Medical Center pathology archive. Clinical data for 121 patients was available. The Armed Forces Institute of Pathology (AFIP) criteria (Miettinen's criteria) was used to risk stratify GISTs using the size, site, and mitotic index of the primary tumor. Immunohistochemistry for CtBP2 and CD44 was then performed on GIST samples with adequate tumor (86, CD44; 87, CtBP2). Stains were scored 0 (negative) to 3 (maximum) by two independent pathologists. Statistical analysis (χ2) correlating AFIP risk category with CD44 and CtBP2 staining was performed using PRISM6. Results: Moderate and high risk GISTs, based on AFIP criteria, significantly correlated with high CtBP2 expression (score = 3, p = 0.046) and low CD44 expression (score = 0-2, p = 0.034). Conclusions: CD44 and CtBP2 staining can be used to risk stratify GIST, and may enhance and complement current clinical risk stratification systems. Our data also suggests a potential tumor suppressive role of CD44 in GIST. Additionally, based on our findings, investigation of therapeutics that target CtBP2 in GISTs should be pursued.

Analysis of copy number loss of the ErbB4 receptor tyrosine kinase in glioblastoma.

Current treatments for glioblastoma multiforme (GBM)—an aggressive form of brain cancer—are minimally effective and yield a median survival of 14.6 months and a two-year survival rate of 30%. Given the severity of GBM and the limitations of its treatment, there is a need for the discovery of novel drug targets for GBM and more personalized treatment approaches based on the characteristics of an individual’s tumor. Most receptor tyrosine kinases—such as EGFR—act as oncogenes, but publicly available data from the Cancer Cell Line Encyclopedia (CCLE) indicates copy number loss in the ERBB4 RTK gene across dozens of GBM cell lines, suggesting a potential tumor suppressor role. This loss is mutually exclusive with loss of its cognate ligand NRG1 in CCLE as well, more strongly suggesting a functional role. The availability of higher resolution copy number data from clinical GBM patients in The Cancer Genome Atlas (TCGA) revealed that a region in Intron 1 of the ERBB4 gene was deleted in 69.1% of tumor samples harboring ERBB4 copy number loss; however, it was also found to be deleted in the matched normal tissue samples from these GBM patients (n = 81). Using the DECIPHER Genome Browser, we also discovered that this mutation occurs at approximately the same frequency in the general population as it does in the disease population. We conclude from these results that this loss in Intron 1 of the ERBB4 gene is neither a de novo driver mutation nor a predisposing factor to GBM, despite the indications from CCLE. A biological role of this significantly occurring genetic alteration is still unknown. While this is a negative result, the broader conclusion is that while copy number data from large cell line-based data repositories may yield compelling hypotheses, careful follow up with higher resolution copy number assays, patient data, and general population analyses are essential to codify initial hypotheses prior to investing experimental resources.

Tumor-suppressive function of UNC5D in papillary thyroid cancer.

BackgroundStudies have shown an association of the UNC5D gene with kidney and bladder cancer and neuroblastoma. We investigated whether UNC5D acts as a tumor suppressor in papillary thyroid carcinoma (PTC).MethodsPrimary PTC tumors and matched normal thyroid tissues were obtained from 112 patients to detect UNC5D mRNA by real-time PCR. Genomic DNA sequencing was performed to detect BRAF mutation in PTC tumors. The association between UNC5D expression and clinicopathological data from PTC patients was reviewed retrospectively. PTC-derived cancer cell lines TPC-1 and K1 with stable transfection of UNC5D were used to investigate the functions of UNC5D. Flow cytometry, CCK-8, Transwell assay and scratch tests were used to examine cell cycle distribution, proliferation and migration.ResultsThe expression of UNC5D was significantly decreased in PTC compared with adjacent normal thyroid tissues. Lower UNC5D expression was significantly associated with aggressive tumor behaviors, such as lymph node metastasis and BRAF mutation. Overexpression of UNC5D significantly suppressed malignant cell behaviors, including cell proliferation and migration, as well as tumor growth in vivo.ConclusionsThese findings suggest a potential tumor suppressor role of UNC5D in PTC progression; and provide insight into potential clinical relevance for the prognosis of PTC.

MIER3 suppresses colorectal cancer progression by down-regulating Sp1, inhibiting epithelial-mesenchymal transition

Mesoderm induction early response 1, family member 3 (MIER3) has recently been identified as a potential cancer susceptibility gene. However, the expression pattern and the role of MIER3 in the progression of colorectal cancer (CRC) have not yet been well characterized. Here, we reported that MIER3 was significantly reduced in human primary colorectal cancer and was associated with CRC metastasis and poor prognosis. Moreover, the up-regulation of MIER3 expression significantly inhibited CRC cell proliferation, migration and invasion in vitro and repressed tumor growth and metastasis in vivo. In contrast, down-regulation of MIER3 could promote the aggressive behaviors of CRC cells. Furthermore, our study showed that MIER3 inhibited cell proliferation and invasion partially via reduction of Sp1 and subsequent suppression of epithelial-mesenchymal transition (EMT). In conclusion, our data suggested that MIER3 plays a potential tumor suppressor role in CRC progression and may be a potentially valuable clinical prognostic marker of this disease.

Abstract 3045: Engineering Mesenchymal Stem Cells (MSCs) to support tumor-targeted delivery of exosome-encapsulated microRNA-379

Abstract Mesenchymal Stem Cells (MSCs) are multipotent stromal cells known to migrate specifically to the sites of tumors and metastases, raising potential as tumor-targeted delivery vehicles for therapeutic agents. MSCs secrete exosomes containing genetic material including microRNAs (miRs), that are effectively taken up by cells. Previous work has shown miR-379 to be significantly reduced in breast tumor tissue, highlighting a potential tumor suppressor role. This study aimed to establish a mechanism of action for miR-379, and to engineer MSCs to secrete exosomes enriched with the miR for tumor-targeted delivery. Methods: RNA was isolated from breast tumors and matching lymph node metastases from the same patients, and miR-379 expression quantified by RQ-PCR. HCC1954 breast cancer cells were transduced with lentivirus to express elevated miR-379 (HCC-379) or a control sequence (HCC-NTC). Cells were implanted into the mammary fat pad (MFP) of mice and tumor progression monitored. Subsequently, MSCs were transduced to generate MSC-379 and MSC-NTC. MSC-secreted exosomes were isolated by differential centrifugation, microfiltration and ultracentrifugation. The morphology, size and number of isolated exosomes was characterized using Transmission Electron Microscopy, Western Blot and Nanoparticle Tracking Analysis respectively. Exosomal miRs were analysed by RQ-PCR. MFP tumors were established using HCC1954-luciferase cells, followed by IV injection of MSC-379 or MSC-NTC cells, or sequential doses of exosomes derived from either cell population. Results: miR-379 expression was significantly reduced in lymph node metastases compared to primary tumors from the same patients (p=0.009), supporting a tumor suppressor role. Analysis of HCC-379 and HCC-NTC tumor growth In Vivo showed no difference in tumor size, however an increase in tumour necrosis (5-50%) and decrease in lymph node invasion was observed in HCC-379 tumors. Investigation of a potential role for miR-379 in regulating COX-2, revealed an inverse relationship (r = -0.48, p=0.02) at a mRNA level, further confirmed at a protein level. MSC-379 cells showed no significant change in migratory or proliferative capacity In Vitro. MSC-secreted exosomes were confirmed to be the correct morphology and size (30-120nm), and to express the exosome-associated protein CD63. An increase in miR-379 (>5 fold) was observed in exosomes secreted by MSC-379 compared to MSC-NTC cells. Administration of MSC-379 or MSC-NTC cells, or exosomes derived from either cell population, was well tolerated In Vivo with no adverse effects observed. Monitoring tumor response to therapy using IVIS is ongoing. Conclusion: The data presented supports miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Engineering tumor-targeted MSCs to secrete exosomes enriched with miR-379 holds exciting potential as a novel therapy for breast cancer. Citation Format: Killian P. O'Brien, Katie Gilligan, Sonja Khan, Brian Moloney, Kerry Thompson, Pierce Lalor, Peter Dockery, Helen Ingoldsby, Michael J. Kerin, Roisin M. Dwyer. Engineering Mesenchymal Stem Cells (MSCs) to support tumor-targeted delivery of exosome-encapsulated microRNA-379 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3045. doi:10.1158/1538-7445.AM2017-3045

Abstract B26: Altered eIF3f subcellular localization and expression in pancreatic ductal adenocarcinomas and its precursor lesions

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the US. The lack of specific diagnostic and prognostic biomarkers is a major obstacle in patient management. Pathologically, chronic pancreatitis can be sometimes difficult to distinguish from pancreatic ductal adenocarcinoma (PDA). Our group was the first to demonstrate the loss of the f subunit of eukaryotic initiation factor 3 (eIF3f) in many types of cancer cells including PDA cells, suggesting the potential tumor suppressive role of eIF3f. Our further studies showed that knockdown of eIF3f expression in normal pancreatic ductal epithelial cells leads to their malignant transformation-confirming the tumor suppressive function of eIF3f. However, the extent of the loss of eIF3f expression in clinical PDA and its precursor lesions (pancreatic intraepithelial neoplasia [PanIN], intraductal papillary mucinous neoplasm [IPMN], mucinous cystic neoplasm [MCN]), and its diagnostic utility is not known. We hypothesize that eIF3f expression is decreased or lost in clinical PDA and its precursor lesions, and the loss of eIF3f expression can be diagnostically useful to differentiate neoplasms from benign reactive ducts. We evaluated the protein expression level of eIF3f using tissue microarrays (TMAs) composed of a total of 49 duplicated cases of human benign pancreatic tissues (n=11), PDA precursor lesions (PanIN, IPMN, MCN, n=18), and PDA (n=20) from Pathology archive, followed by immunohistochemistry study of eIF3f. We then assessed the subcellular localization, intensity of the IHC staining, and percentage of positive cells by a gastrointestinal pathologist using light microscopy and correlated the findings with the clinical diagnosis. We also compared the staining pattern among the 3 most common IPMN subtypes defined by epithelial cell lineage (gastric, intestinal and pancreatobiliary subtypes). Fisher’s exact tests were used to test the association. A generalized linear regression model with a logit link was used to compare different diagnoses. ANOVA models were used to compare the continuous scores (intensity*percentage) across different diagnoses. All analyses were conducted using SAS (version 9.4, SAS Institute, Cary, NC). Interestingly, we found that there is a significant difference between the eIF3f staining pattern in benign pancreatic tissue, PDA precursor lesions, and PDA. In benign pancreatic ducts, eIF3f was exclusively located in the nuclei and none were located in the cytoplasm (0%). However, significantly increasing percentages of PDA and PDA precursor cases have cytoplasmic eIF3f staining (28% precursor lesions and 45% PDA, p<0.001). Furthermore, there is a significant loss of eIF3f staining in PDA and PDA precursor lesions (5.6% precursor lesion and 40% PDA are negative for eIF3f) compared to benign pancreatic ductal epithelium (all retained eIF3f staining, p<0.01). The diagnostic sensitivity and specificity of eIF3f are 60.5% and 100% respectively. The other interesting finding is that eIF3f is most frequently located in the cytoplasm or lost in the gastric type IPMN which was thought to have the most benign behavior compared to intestinal and pancreatobiliary type IPMN. In summary, we found altered subcellular localization and expression of eIF3f protein in not only PDA, but also PDA precursor lesions, suggesting its important role in PDA development. eIF3f is a highly specific biomarker for the diagnosis of PDA and its precursor lesions. These findings are significant because eIF3f can be a useful diagnostic biomarker to distinguish benign reactive pancreatic ducts from dysplastic or neoplastic pancreatic ductal lesions and a potential prognostic marker in pancreatic neoplasms. Citation Format: Jiaqi Shi, Brittany Silverman, Lili Zhao.{Authors}. Altered eIF3f subcellular localization and expression in pancreatic ductal adenocarcinomas and its precursor lesions. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B26.

Critical Function of PRDM2 in the Neoplastic Growth of Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) derive from primordial germ cells. Their maturation is blocked at different stages, reflecting histological tumor subtypes. A common genetic alteration in TGCT is a deletion of the chromosome 1 short arm, where the PRDM2 gene, belonging to the Positive Regulatory domain gene (PRDM) family, is located. Expression of PRDM2 gene is shifted in different human tumors, where the expression of the two principal protein forms coded by PRDM2 gene, RIZ1 and RIZ2, is frequently unbalanced. Therefore, PRDM2 is actually considered a candidate tumor suppressor gene in different types of cancer. Although recent studies have demonstrated that PRDM gene family members have a pivotal role during the early stages of testicular development, no information are actually available on the involvement of these genes in TGCTs. In this article we show by qRT-PCR analysis that PRDM2 expression level is modulated by proliferation and differentiation agents such as estradiol, whose exposure during fetal life is probably an important risk factor for TGCTs development in adulthood. Furthermore in normal and cancer germ cell lines, PRDM2 binds estradiol receptor α (ERα) and influences proliferation, survival and apoptosis, as previously reported using MCF-7 breast cancer cell line, suggesting a potential tumor-suppressor role in TGCT formation.

GRK2 Is a Novel Modulator of the MALT1 Oncoprotein

Background and Significance: Lymphocyte antigen receptor-dependent stimulation of the NF-κB transcription factor is a key event during the normal adaptive immune response. However, dysregulated activation of these signaling pathways can lead to lymphoma. Stimulation of the B-or T-cell receptor promotes assembly of the CARMA1-Bcl10-MALT1 (CBM) signaling complex, where MALT1 functions as the essential effector molecule by carrying out two important functions: First, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, acts as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of canonical NF-κB signaling. Activating mutations in either BCR or CARMA1 promote constitutive and exaggerated MALT1 activity and are seen as driver mutations in diffuse large B cell lymphoma (DLBCL). Aberrant MALT1 activity also underlies the pathogenesis of MALT lymphoma, another subtype of B-cell malignancy. As such, MALT1 has emerged as a potential new target for pharmaceutical inhibition in the treatment of lymphoma. Here, we describe our discovery of a novel MALT1-binding partner, G protein-coupled receptor kinase 2 (GRK2). We hypothesize that GRK2 plays a critical role in the pathogenesis of lymphoma by modulating MALT1 activity.Methods and Results: Using co-immunoprecipitation and liquid chromatography mass spectrometry we identified GRK2 as a novel MALT1-interacting protein. We validated our results by showing that endogenous GRK2 and MALT1 co-immunoprecipitate (CO-IP) in both B cells and T cells. We next utilized GST-tagged purified proteins to demonstrate a direct interaction between GRK2 and MALT1. We then performed a number of studies to investigate the functional consequences of GRK2 interaction with MALT1. A CO-IP assay demonstrated that GRK2 inhibits Bcl10-dependent binding to MALT1, and a luciferase reporter assay revealed that GRK2 inhibits Bcl10/MALT1-dependent NF-κB activity. In addition, we demonstrated that GRK2 also inhibits MALT1 proteolytic activity using Western blot analysis of two known MALT1 substrates, the deubiquitinase cylindromatosis (CYLD) and the NF-kB family member RelB. Moreover, in Jurkat T cells, overexpression of GRK2 impeded AgR-induced NF-κB activation while stable GRK2 knockdown resulted in enhanced AgR-induced phosphorylation of inhibitor of kappaB (IκB) and secretion of interleukin2 (IL-2).We next began to investigate the potential of GRK2 to function as a tumor suppressor by blocking MALT1 activity. Of note, utilizing a published data set to compare GRK2 mRNA expression in 44 DLBCL patient samples to that of 20 samples of healthy B-cell controls, we found that GRK2 mRNA levels are markedly lower in DLBCL cases. We also compared GRK2 mRNA levels among patients diagnosed with DLBCL grouped by vital status, using data from the Cancer Genome Atlas (TCGA). We found that tumors from patients who died of disease within three years expressed significantly lower levels of GRK2 as compared to tumors from patients alive after three years. We also wished to investigate the impact of GRK2 expression on activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) cells, which require MALT1 activity for growth and survival. We were successful in engineering GRK2 overexpression in control germinal center B-cell like (GCB) DLBCL cells, which do not depend on MALT1 activity for growth. In contrast, we found that in ABC-DLBCL cells, which do depend on MALT1 activity, GRK2 overexpression greatly impairs cell proliferation, making it difficult to grow these engineered cells. These data provide support for the concept that GRK2 may have a potential tumor suppressor role in MALT1-dependent lymphoma.Conclusions: Our study represents the first report of a negative modulator of MALT1 and provides critical new insight into mechanisms regulating its activity. We hope to harness this information in developing novel strategies for inhibiting MALT1-dependent lymphomagenesis. DisclosuresNo relevant conflicts of interest to declare.

ZNRF3 is downregulated in papillary thyroid carcinoma and suppresses the proliferation and invasion of papillary thyroid cancer cells.

Zinc and ring finger 3 (ZNRF3) is a transmembrane E3 ubiquitin ligase that has emerged as an important regulator of cancer development; however, its cancer-related function remains controversial. Here, we investigated the possible role of ZNRF3 in thyroid carcinoma (TC). We found that ZNRF3 is downregulated in papillary thyroid carcinoma (PTC) compared to normal thyroid tissues and inversely correlated with the degree of cell differentiation. Overexpression of ZNRF3 significantly suppressed cell malignant behaviors, including cell proliferation, migration, and invasion in vitro, as well as tumor growth in vivo. Consistent with recent studies showing that ZNRF3 is involved in the Wnt/β-catenin pathway, ZNRF3 overexpression negatively regulated β-catenin activation, modulating PTC cell behaviors. Clinical specimens revealed a significant inverse correlation between ZNRF3 and β-catenin mRNA levels. Taken together, these results provide insight into a potential tumor suppressor role of ZNRF3 in PTC progression, and may have potential clinical relevance for the prognosis and treatment of PTC.

Abstract 1158: Differential expression and functional impact of the alternatively spliced transcripts of Extracellular matrix protein 1 in esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in China, with five-year survival rates in Hong Kong being of ∼14%. An oligonucleotide microarray was utilized to identify differentially-expressed genes in ESCC using 31 pairs of matched normal/tumor samples from Hong Kong and Henan, China. Extracellular matrix protein 1 (ECM1) was among the top down-regulated genes, indicating a potential tumor-suppressive role. Public cDNA microarray data also shows significant down-regulation of ECM1 expression in ESCC. Interestingly, this gene has been extensively studied in breast cancer and thyroid cancer for its oncogenic role. There are three alternatively-spliced variants of ECM1. Therefore, this gene was selected for further variant expression and functional analysis in ESCC. Results: RNA sequencing analysis of matched normal/ESCC samples showed that ECM1b (NM_022664) is the dominant expressed variant in esophagus, followed by ECM1a (NM_004425). In tumor samples, the expression of ECM1a is down-regulated and that of ECM1b is down-regulated or lost, as compared to normal counterparts. Expression of ECM1c (NM_001202858) is not detected in either normal or tumor samples. Quantitative PCR using variant-specific primer sets confirms the findings of microarray and RNA sequencing analyses. When re-expressed in ESCC cell lines after lentiviral transduction, ECM1b expression shows a trend of tumor suppression in the orthotopic ESCC mouse tumorigenicity model; ECM1a shows no tumor-suppressive effect in either. ECM1b expression also shows a metastasis inhibitory effect in a tail vein experimental metastasis model. Immunohistochemical staining shows E-cadherin up-regulation in orthotopic tumors re-expressing ECM1b. Conclusions: These results suggest differential roles of variants of ECM1 in ESCC. Unlike the cases in other types of cancer, both expression of ECM1a and ECM1b is down-regulated in ESCC compared to normal esophageal tissues. ECM1b represents the dominantly expressed variants in esophagus epithelium, and its expression is highly reduced in esophageal carcinoma. ECM1b plays a potential suppressive role in tumorigenesis and metastasis. Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for funding support (HKU 774413M to M.L.L). Citation Format: Valen Zhuoyou YU, Josephine Mun-Yee Ko, Simon Law, Li Dong Wang, Maria Li Lung. Differential expression and functional impact of the alternatively spliced transcripts of Extracellular matrix protein 1 in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1158.

Abstract 946: Epigenetic silencing of tumor suppressor long non-coding RNA BM742401 in chronic lymphocytic leukemia

Abstract Background: B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. Long non-coding RNAs (lncRNAs) emerge as a new class of ncRNAs measuring longer than 200 nucleotides in length, and implicated in a wide range of biological processes including normal development, differentiation or carcinogenesis [1]. Recently, deregulated expression of lncRNAs has been found in many human cancers, indicating its potential oncogenic or tumor suppressor role in carcinogenesis [2]. However, data on the role of lncRNA in CLL is scanty. BM742401 is a tumor suppressor lncRNA downregulated in gastric cancer [3]. As the promoter region and the entire transcript are embedded in a CpG island, we postulated that BM742401 is a tumor suppressor lncRNA inactivated by DNA methylation in CLL. Methods: Methylation of BM742401 was studied by methylation-specific polymerase chain reaction (MSP) in nine healthy donor normal controls including three bone marrow, three peripheral blood buffy coats, and three CD19-sorted peripheral B-cells, in addition to seven CLL cell lines, and ninety-eight diagnostic CLL marrow samples. Results: By MSP, promoter of BM742401 was unmethylated in normal controls, but methylated in four out of seven (57%) CLL cell lines. MSP statuses, including complete methylation (MM), partial methylation (MU), and complete unmethylation (UU), were verified by quantitative bisulfite pyrosequencing. Methylation of BM742401 was inversely correlated with expression. In the completely methylated WAC3CD5+ CLL cells, treatment with 5-Aza-2’-deoxycytidine led to promoter demethylation and re-expression of BM742401 transcript. Functionally, stable overexpression of BM742401 by lentiviral transduction resulted in inhibition of cellular proliferation and enhanced apoptosis through caspase-9-dependent intrinsic apoptosis pathway, suggesting a tumor suppressor role of BM742401 in CLL. In primary CLL samples, methylation of BM742401 was detected in 43/98 (44%) of patients. Moreover, among CLL patients with standard-risk cytogenetic aberrations, methylation of BM742401 correlated with advanced Rai stage (≥ stage 2) (P = 0.002). Furthermore, methylation of BM742401 was significantly associated with methylation of miR-129-2 (P = 0.034). Conclusions: BM742401 is a tumor suppressor lncRNA frequently methylated in CLL. The functional mechanism of BM742401 in triggering tumor suppressive phenotype and regulation of other protein-coding genes warrant further studies.

TP53 Mutations in Head and Neck Squamous Cell Carcinoma and Their Impact on Disease Progression and Treatment Response.

Recent studies describing the mutational landscape of head and neck squamous cell carcinoma (HNSCC) on a genomic scale by our group and others, including The Cancer Genome Atlas, have provided unprecedented perspective for understanding the molecular pathogenesis of HNSCC progression and response to treatment. These studies confirmed that mutations of the TP53 tumor suppressor gene were the most frequent of all somatic genomic alterations in HNSCC, alluding to the importance of the TP53 gene in suppressing the development and progression of this disease. Clinically, TP53 mutations are significantly associated with short survival time and tumor resistance to radiotherapy and chemotherapy in HNSCC patients, which makes the TP53 mutation status a potentially useful molecular factor for risk stratification and predictor of clinical response in these patients. In addition to loss of wild-type p53 function and the dominant-negative effect on the remaining wild-type p53, some p53 mutants often gain oncogenic functions to promote tumorigenesis and progression. Different p53 mutants may possess different gain-of-function properties. Herein, we review the most up-to-date information about TP53 mutations available via The Cancer Genome Atlas-based analysis of HNSCC and discuss our current understanding of the potential tumor-suppressive role of p53, focusing on gain-of-function activities of p53 mutations. We also summarize our knowledge regarding the use of the TP53 mutation status as a potential evaluation or stratification biomarker for prognosis and a predictor of clinical response to radiotherapy and chemotherapy in HNSCC patients. Finally, we discuss possible strategies for targeting HNSCCs bearing TP53 mutations. J. Cell. Biochem. 117: 2682-2692, 2016. © 2016 Wiley Periodicals, Inc.

JNK1/2 expression and modulation of STAT3 signaling in oral cancer.

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that link extracellular stimuli with intracellular responses and participate in numerous cellular processes such as growth, proliferation, differentiation, inflammation and apoptosis. Persistent activation of signal transducer and activator of transcription 3 (STAT3), which is accompanied by increases in STAT3 tyrosine phosphorylation, is associated with cell proliferation, differentiation and apoptosis in oral squamous cell carcinoma (OSCC). The role and significance of the activation of MAPKs, particularly of c-Jun N-terminal kinase (JNK), on STAT3 signaling in OSCC have not been thoroughly investigated. The present study examines the effects of JNK1/2 modulation on STAT3 signaling and cellular activities in OSCC cells. The expression levels of STAT3 [total, tyrosine phosphorylated (p-Tyr) and serine phosphorylated (p-Ser)], JNK, c-Jun and cyclin D1 were assessed in the OSCC cell lines SCC25 and SCC9. Inhibition of JNK1/2 was achieved by pharmacological agents (SP600125) and by small interfering RNA (siRNA) silencing, while JNK1/2 was induced by active MAPK kinase 7. Cell proliferation and viability rates were also evaluated. Inhibition of JNK1/2 with either SP600125 treatment or specific siRNA silencing resulted in decreased levels of p-Ser STAT3 and increased levels of p-Tyr STAT3 and cyclin D1 in both cell lines. Furthermore, JNK1/2 inhibition resulted in a dose-dependent increase in cell growth and viability in both cell lines. Opposite results were observed with JNK1/2 induction in both cell lines. The present results are supportive of a potential tumor suppressive role of JNK1/2 signaling in OSCC, which may be mediated through negative crosstalk with the oncogenic STAT3 signaling pathway. The possible therapeutic implications of JNK1/2 inhibition for patients with OSCC require to be investigated.