Hong Lok Lung

Tumor suppressor Alpha B-crystallin ( CRYAB ) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties

Alpha B-crystallin (CRYAB) maps within the nasopharyngeal carcinoma (NPC) tumor-suppressive critical region 11q22-23 and its downregulation is significantly associated with the progression of NPC. However, little is known about the functional impact of CRYAB on NPC progression. In this study we evaluated the NPC tumor-suppressive and progression-associated functions of CRYAB. Activation of CRYAB suppressed NPC tumor formation in nude mice. Overexpression of CRYAB affected NPC progression-associated phenotypes such as loss of cell adhesion, invasion, interaction with the tumor microenvironment, invasive protrusion formation in three dimensional Matrigel culture, as well as expression of epithelial–mesenchymal transition-associated markers. CRYAB mediates this ability to suppress cancer progression by inhibition of E-cadherin cytoplasmic internalization and maintenance of β-catenin in the membrane that subsequently reduces the levels of expression of critical downstream targets such as cyclin-D1 and c-myc. Both ectopically expressed and recombinant CRYAB proteins were associated with endogenous E-cadherin and β-catenin, and, thus, the cadherin/catenin adherens junction. The CRYAB α-crystallin core domain is responsible for the interaction of CRYAB with both E-cadherin and β-catenin. Taken together, these results indicate that CRYAB functions to suppress NPC progression by associating with the cadherin/catenin adherens junction and modulating the β-catenin function.

Nuclear Localization of DNAJB6 Is Associated With Survival of Patients With Esophageal Cancer and Reduces AKT Signaling and Proliferation of Cancer Cells

BACKGROUND & AIMSnThe DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6) is part of a family of proteins that regulates chaperone activities. One of its isoforms, DNAJB6a, contains a nuclear localization signal and regulates β-catenin signaling during breast cancer development. We investigated the role of DNAJB6 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).nnnMETHODSnWe performed immunohistochemical analyses of primary ESCC samples and lymph node metastases from a cohort of 160 patients who underwent esophagectomy with no preoperative chemoradiotherapy at Hong Kong Queen Mary Hospital. Data were collected on patient outcomes over a median time of 12.1 ± 2.9 months. Retrospective survival association analyses were performed. Wild-type and mutant forms of DNAJB6a were overexpressed in cancer cell lines (KYSE510, KYSE 30TSI, KYSE140, and KYSE70TS), which were analyzed in proliferation and immunoblot assays, or injected subcutaneously into nude mice. Levels of DNAJB6 were knocked down in ESCC cell lines (KYSE450 and T.Tn), immortalized normal esophageal epithelial cell lines (NE3 and NE083), and other cells with short hairpin RNAs, or by genome engineering. Bimolecular fluorescence complementation was used to study interactions between proteins in living cells.nnnRESULTSnIn primary ESCC samples, patients whose tumors had high nuclear levels of DNAJB6 had longer overall survival times (19.2 ± 1.8 months; 95% confidence interval [CI], 15.6-22.8 mo) than patients whose tumors had low nuclear levels of DNAJB6 (12.6 ± 1.4 mo; 95% CI, 9.8-15.4 mo; Pxa0= .004, log-rank test). Based on Cox regression analysis, patients whose tumors had high nuclear levels of DNAJB6 had a lower risk of death than patients with low levels (hazard ratio, 0.562; 95% CI, 0.379-0.834; Pxa0= .004). Based on log-rank analysis and Cox regression analysis, the combination of the nuclear level of DNAJB6 and the presence of lymph node metastases at diagnosis could be used to stratify patients into groups with good or bad outcomes (P < .0005 for both analyses). There was a negative association between the nuclear level of DNAJB6 and the presence of lymph node metastases (Pxa0= .022; Pearson χ(2) test). Cancer cell lines that overexpressed DNAJB6a formed tumors more slowly in nude mice than control cells or cells that expressed a mutant form of DNAJB6a that did not localize to the nucleus. DNAJB6 knockdown in cancer cell lines promoted their growth as xenograft tumors in mice. A motif of histidine, proline, and aspartic acid in the J domain of DNAJB6a was required for its tumor-suppressive effects and signaling via AKT1. Loss of DNAJB6a resulted in up-regulation of AKT signaling in cancer cell lines and immortalized esophageal epithelial cells. Expression of a constitutively active form of AKT1 restored proliferation to tumor cells that overexpressed DNAJB6a, and DNAJB6a formed a complex with AKT1 in living cells. The expression of DNAJB6a reduced the sensitivity of ESCC to AKT inhibitors; the expression level of DNAJB6a affected AKT signaling in multiple cancer cell lines.nnnCONCLUSIONSnNuclear localization of DNAJB6 is associated with longer survival times of patients with ESCC. DNAJB6a reduces AKT signaling, and DNAJB6 expression in cancer cells reduces their proliferation and growth of xenograft tumors in mice. DNAJB6a might be developed as a biomarker for progression of ESCC.

Significance of NF-κB activation in immortalization of nasopharyngeal epithelial cells.

NF‐κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF‐κB is common in NPC and may contribute to NPC development. The role of NF‐κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF‐κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF‐κB signaling is a common phenotype in telomerase‐immortalized NPE cell lines. Our results suggest that NF‐κB activation promotes the growth of telomerase‐immortalized NPE cells, and suppression of NF‐κB activity inhibits their proliferation. Furthermore, we observed upregulation of c‐Myc, IL‐6 and Bmi‐1 in our immortalized NPE cells. Inhibition of NF‐κB downregulated expression of c‐Myc, IL‐6 and Bmi‐1, suggesting that they are downstream events of NF‐κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF‐κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF‐κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.

NF-κB p65 Subunit Is Modulated by Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) in Nasopharyngeal Carcinoma HONE1 and HK1 Cells

NF-κB is a well-characterized transcription factor, widely known as a key player in tumor-derived inflammation and cancer development. Herein, we present the functional and molecular relevance of the canonical NF-κB p65 subunit in nasopharyngeal carcinoma (NPC). Loss- and gain-of-function approaches were utilized to reveal the functional characteristics of p65 in propagating tumor growth, tumor-associated angiogenesis, and epithelial-to-mesenchymal transition in NPC cells. Extracellular inflammatory stimuli are critical factors that trigger the NF-κB p65 signaling; hence, we investigated the components of the tumor microenvironment that might potentially influence the p65 signaling pathway. This led to the identification of an extracellular matrix (ECM) protein that was previously reported as a candidate tumor suppressor in NPC. Our studies on the Latent Transforming Growth Factor-β Binding Protein 2 (LTBP2) protein provides substantial evidence that it can modulate the p65 transcriptional activity. Re-expression of LTBP2 elicits tumor suppressive effects that parallel the inactivation of p65 in NPC cells. LTBP2 was able to reduce phosphorylation of p65 at Serine 536, inhibit nuclear localization of active phosphorylated p65, and impair the p65 DNA-binding ability. This results in a consequential down-regulation of p65-related gene expression. Therefore, the data suggest that the overall up-regulation of p65 expression and the loss of this candidate ECM tumor suppressor are milestone events contributing to NPC development.

SAA1 polymorphisms are associated with variation in antiangiogenic and tumor-suppressive activities in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a cancer that occurs in high frequency in Southern China. A previous functional complementation approach and the subsequent cDNA microarray analysis have identified that serum amyloid A1 (SAA1) is an NPC candidate tumor suppressor gene. SAA1 belongs to a family of acute-phase proteins that are encoded by five polymorphic coding alleles. The SAA1 genotyping results showed that only three SAA1 isoforms (SAA1.1, 1.3 and 1.5) were observed in both Hong Kong NPC patients and healthy individuals. This study aims to determine the functional role of SAA1 polymorphisms in tumor progression and to investigate the relationship between SAA1 polymorphisms and NPC risk. Indeed, we have shown that restoration of SAA1.1 and 1.3 in the SAA1-deficient NPC cell lines could suppress tumor formation and angiogenesis in vitro and in vivo. The secreted SAA1.1 and SAA1.3 proteins can block cell adhesion and induce apoptosis in the vascular endothelial cells. In contrast, the SAA1.5 cannot induce apoptosis or inhibit angiogenesis because of its weaker binding affinity to αVβ3 integrin. This can explain why SAA1.5 has no tumor-suppressive effects. Furthermore, the NPC tumors with this particular SAA1.5/1.5 genotype showed higher levels of SAA1 gene expression, and SAA1.1 and 1.3 alleles were preferentially inactivated in tumor tissues that were examined. These findings further strengthen the conclusion for the defective function of SAA1.5 in suppression of tumor formation and angiogenesis. Interestingly, the frequency of the SAA1.5/1.5 genotype in NPC patients was ~2-fold higher than in the healthy individuals (P=0.00128, odds ratio=2.28), which indicates that this SAA1 genotype is significantly associated with a higher NPC risk. Collectively, this homozygous SAA1.5/1.5 genotype appears to be a recessive susceptibility gene, which has lost the antiangiogenic function, whereas SAA1.1 and SAA1.3 are the dominant alleles of the tumor suppressor phenotype.

Novel lentiviral-inducible transgene expression systems and versatile single-plasmid reporters for in vitro and in vivo cancer biology studies.

Many of the cancer cell lines derived from solid tumors are difficult to transfect using commonly established transfection approaches. This hurdle for some DNA transfection systems has hindered cancer biology studies. Moreover, there are limited tools for studying pathway activities. Therefore, highly efficient improved gene transfer and versatile genetic tools are required. In this study, we established and developed a comprehensive set of new lentiviral tools to study gene functions and pathway activities. Using the optimized conditions, cancer cell lines achieved >90% transduction efficiency. Novel lentiviral doxycycline-regulated pTet-IRES-EGFP (pTIE) systems for transgene expression and TRE reporters used for pathway activity determination were developed and tested. The pTIE Tet-Off system showed in vitro doxycycline-sensitive responses with low or undetectable leakage of protein expression and in vivo tumor suppression as illustrated using candidate tumor suppressors, Fibulin-2 and THY1. In contrast, the Tet-On system showed dose-dependent responses. The pTRE-EGFP (pTE) and pTRE-FLuc-EF1α-RLuc (pT-FER) reporters with the NFκB p65 subunit consensus sequence showed GFP and firefly luciferase responses, which were directly correlated with TNFα stimulation, respectively. Taken together, these newly developed lentiviral systems provide versatile in vitro and in vivo platforms to strengthen our capabilities for cancer biology studies.

Roles of Tumor Suppressor Signaling on Reprogramming and Stemness Transition in Somatic Cells

The pioneering landmark, established by Takahashi and Yamanaka (Takahashi et al., 2007; Takahashi and Yamanaka, 2006) in reprogramming somatic cells into induced pluripotent stem (iPS) cells using the four transcriptional factors of Oct4, Sox2, Klf4, and c-Myc, represents one of the most important paradigm shifts in current stem cell biology. This unprecedented discovery could potentially revolutionize regenerative medicine, cell-based therapy and personalized medicine. Despite recent great advancement in cell reprogramming, there are still considerable technical challenges to circumvent restrictions of applications of reprogram‐ ming technology (Kawamura et al., 2009; Saha and Jaenisch, 2009). The utilization of overexpressed transcriptional factors, which of many play oncogenic roles, during somatic reprogramming posts the risk of malignant transformation, thus, limiting its clinical applica‐ tions. Moreover, the reprogramming process using these factors is still inefficient in some of cell types, and is not always successful in other kinds of cells (Kawamura et al., 2009; Marion et al., 2009; Menendez et al., 2012). Therefore, the underlying mechanisms for signaling control of these factors still need to be further explored.

Identification of Tumor Suppressor Genes via Cell Fusion and Chromosomal Transfer

Loss of DNA or chromosomal deletion was frequently reported in different sporadic tumors, suggesting that those lost regions may contain putative tumor suppressor genes (TSGs). To map and isolate candidate genes from vast randomly lost areas, functional and complementary evidence is usually needed to define these areas to critical regions (CR). This is particularly important when one is dealing with sporadic cancers where clearly defined familial predisposition is present but high cancer risk families are not available for position cloning. A numerous studies have revealed extensive DNA deletions in nasopharyngeal carcinoma (NPC) and esophageal squamous cell carcinoma (ESCC). To identify candidate genes from these cancers, we used cell fusion and microcell-mediated chromosome transfer (MMCT) to introduce the whole chromosome or a chromosome fragment, into NPC and ESCC cell lines. Combined with other molecular approaches, we have successfully identified a number of novel TSGs on various human chromosomes. The nasopharynx is located behind the nasal cavity in the upper part of the pharynx (Fig. 1). NPC is a type of malignancy which arises from the epithelial cells in the nasopharynx. NPC is an unique cancer, which is commonly found in, Southeast Asia, North Africa, Middle East, and Arctic regions, but rare in most parts of the world (Jeyakumar et al 2006, Wei et al 2005). The esophagus is a tube, which is about 25 cm long, for the food passage from mouth to stomach (Fig. 2). Esophageal cancer (EC) is classified into two major histologic subtypes: squamous cell carcinoma and adenocarcinoma (Daly et al 2000). Esophageal adenocarcinoma (EAC) arises from the cells of glands responsible for producing mucous in the esophageal wall. The majority of the cases (>80%) are ESCC in Hong Kong, while EAC shows a climbing incidence in Western countries. EC varies greatly in geographical distribution; high-risk areas include north-central China in Henan and Shanxi (Holmes et al 2007, Qi et al 2005, Wu et al 2006).