Michelle L.Y. Wong

The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation

Identification of tumor suppressor genes (TSG) silenced by methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic tumor markers for early cancer detection. Both nasopharyngeal carcinoma (NPC) and esophageal carcinoma are major tumors in Southern China and Southeast Asia. Through expression subtraction of NPC, we identified Deleted in Liver Cancer 1 (DLC1)/ARHGAP7 (NM_006094) – an 8p22 TSG as a major downregulated gene. Although expressed in all normal tissues, DLC1 was silenced or downregulated in 11/12 (91%) NPC, 6/15 (40%) esophageal, 5/8 (63%) cervical and 3/9 (33%) breast carcinoma cell lines. No genetic deletion of DLC1 was detected in NPC although a hemizygous deletion at 8p22–11 was found by 1-Mb array-CGH in some cell lines. We then located the functional DLC1 promoter by 5′-RACE and promoter activity assays. This promoter was frequently methylated in all downregulated cell lines and in a large collection of primary tumors including 89% (64/72) NPC (endemic and sporadic types), 51% (48/94) esophageal, 87% (7/8) cervical and 36% (5/14) breast carcinomas, but seldom in paired surgical marginal tissues and not in any normal epithelial tissue. The transcriptional silencing of DLC1 could be reversed by 5-aza-2′-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B. Furthermore, ectopic expression of DLC1 in NPC and esophageal carcinoma cells strongly inhibited their colony formation. We thus found frequent epigenetic silencing of DLC1 in NPC, esophageal and cervical carcinomas, and a high correlation of methylation with its downregulation, suggesting a predominant role of epigenetic inactivation. DLC1 appears to be a major TSG implicated in the pathogenesis of these tumors, and should be further tested as a molecular biomarker in patients with these cancers.

Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis

Background: The antiapoptotic and epithelial–mesenchymal transition activities of Twist have been implicated in the neoplastic transformation and the development of metastasis, respectively. Upregulation of Twist, described in several types of human cancer, also acts as a prognostic marker of poor outcome. Aim: To investigate Twist expression in oesophageal squamous cell carcinoma (SCC) and its prognostic value in a Chinese cohort of patients with oesophageal SCC. Methods: Twist expression in primary oesophageal SCC of 87 Chinese patients was investigated by immunohistochemical staining. Twist protein level in one immortalised normal oesophageal epithelial cell line and six oesophageal SCC cell lines was measured by western blot analysis. Twist mRNA level in 30 pairs of frozen specimens of primary oesophageal SCC and non-neoplastic oesophageal epithelium from the upper resection margin of corresponding oesophagectomy specimen was also determined by semiquantitative reverse transcription-PCR. Results: It was found that Twist was upregulated in oesophageal SCC cell lines, and its mRNA and protein levels were both increased in oesophageal SCC and the non-neoplastic oesophageal epithelium (p<0.001). In addition, a high level of Twist expression in oesophageal SCC was significantly associated with a greater risk for the patient of developing distant metastasis within 1 year of oesophagectomy (OR 3.462, 95% CI 1.201 to 9.978; p = 0.022). Conclusions: Our results suggest that upregulation of Twist plays a role in the neoplastic transformation to oesophageal SCC and subsequent development of distant metastasis. Twist may serve as a useful prognostic marker for predicting the development of distant metastasis in oesophageal SCC.

Receptor-type tyrosine-protein phosphatase κ directly targets STAT3 activation for tumor suppression in nasal NK/T-cell lymphoma

Nasal-type natural killer/T-cell lymphoma (NKTCL) is an aggressive disease characterized by frequent deletions on 6q, and constitutive activation of signal transducer and activator of transcription 3 (STAT3). Phosphorylation at Tyr705 activates STAT3, inducing dimerization, nuclear translocation, and DNA binding. In this study, we investigated whether receptor-type tyrosine-protein phosphatase κ (PTPRK), the only protein tyrosine phosphatase at 6q that contains a STAT3-specifying motif, negatively regulates STAT3 activation in NKTCL. PTPRK was highly expressed in normal NK cells but was underexpressed in 4 of 5 (80%) NKTCL cell lines and 15 of 27 (55.6%) primary tumors. Significantly, PTPRK protein expression was inversely correlated with nuclear phospho-STAT3(Tyr705) expression in NKTCL cell lines (P = .025) and tumors (P = .040). PTPRK restoration decreased nuclear phospho-STAT3(Tyr705) levels, whereas knockdown of PTPRK increased such levels in NKTCL cells. Phosphatase substrate-trapping mutant assays demonstrated the binding of PTPRK to STAT3, and phosphatase assays showed that PTPRK directly dephosphorylated phospho-STAT3(Tyr705). Restoration of PTPRK inhibited tumor cell growth and reduced the migration and invasion ability of NKTCL cells. Monoallelic deletion and promoter hypermethylation caused underexpression of PTPRK messenger RNA in NKTCL, and methylation of the PTPRK promoter significantly correlated with inferior overall survival (P = .049) in NKTCL patients treated with the steroid-dexamethasone, methotrexate, ifosfamide, l-asparaginase, and etoposide regimen. Altogether, our findings show that PTPRK underexpression leads to STAT3 activation and contributes to NKTCL pathogenesis.

The Role of Pea3 Group Transcription Factors in Esophageal Squamous Cell Carcinoma

The transcription factors Pea3, Erm, and Er81 can promote cancer initiation and progression in various types of solid tumors. However, their role in esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we found that the expression levels of Pea3 and Erm, but not that of Er81, were significantly higher in ESCC compared with nontumor esophageal epithelium. A high level of Pea3 expression was significantly correlated with a shorter overall survival in a cohort of 81 patients with ESCC and the subgroup with N1 stage tumor (Wilcoxon-Gehan test, P = 0.016 and P = 0.001, respectively). Pea3 was overexpressed in seven ESCC cell lines compared with two immortalized esophageal cell lines. Pea3 knockdown reduced cell proliferation and suppressed nonadherent growth, migration, and invasion in ESCC cells in vitro. In addition, Pea3 knockdown in ESCC cells resulted in a down-regulation of phospho-Akt and matrix metalloproteinase 13, whereas a significant positive correlation in the expression levels was observed between Pea3 and phospho-Akt (r = 0.281, P < 0.013) and between Pea3 and matrix metalloproteinase 13 in the human specimens (r = 0.462, P < 0.001). Moreover, Pea3 modulated the sensitivity of EC109 cells to doxorubicin, probably via reduced activity of the phosphatidylinositol 3-kinase-Akt-mammalian target of Rapamycin complex 1 pathway on Pea3 knockdown. In conclusion, our results suggest that Pea3 plays an important role in the progression of ESCC.

CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHSmu were detected in follicular lymphomas and exclusively in germinal center B cell-ike (GCB)-DLBCLs. CD44 is not expressed in reactive GC B cells. The IGHSmu/CD44 translocations substitute Smu for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Imu-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44DeltaEx1). When overexpressed in vitro in the CD44(-) GCB-DLBCL cell line BJAB, CD44DeltaEx1-green fluorescent protein localized to the cytoplasm and nucleus, whereas CD44s-green fluorescent protein (standard form) localized to the plasma membrane. The ectopic expression of CD44DeltaEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation.

Relative contributions of vowels and consonants in recognizing isolated Mandarin words

Abstract This study investigated the relative contributions of vowels and consonants in recognizing isolated Mandarin words. Normal-hearing native-Mandarin listeners were instructed to recognize isolated Mandarin words and identify consonants, vowels and tones with stimuli synthesized to contain different proportions of consonant or vowel segments, including five conditions of consonant-only, vowel-only, consonant or vowel plus consonant–vowel (C–V) transition, and C–V transition. The recognition score of the vowel-only Mandarin words was significantly higher than that of the consonant-only words; and word recognition scores had a higher correlation with vowel identification scores than consonant identification scores. Moreover, adding a small portion of C–V transition significantly improved the recognition score of the consonant-only Mandarin words. In the conditions of C–V transition and consonant plus C–V transition, the duration of preserved portion predicted modestly well the scores of isolated word recognition, and vowel, consonant and lexical tone identification in Mandarin. These findings suggest that there is a greater contribution of vowels than consonants to isolated word recognition in Mandarin, which is different from previous outcomes in English.

Abstract 258: Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm

Dysregulation of an oncogene by translocation to Ig locus is a common event in the pathogenesis of most non-Hodgkin9s lymphoma. Using inverse-PCR, CD44 on 11p13 was identified as a novel translocation partner of IgH in 9 of 114 cases of gastric, non-gastric extranodal, follicular and nodal diffuse large B-cell lymphomas (DLBCLs) analyzed. IgHSμ/CD44 translocation juxtaposes the enhancer of IgHSμ to the 5’ regulatory region of CD44 in a tail-to-head orientation, leading to the removal of exon 1 of CD44. Sequencing analysis showed microhomology sequences at the junction breakpoints of all the IGHSμ/CD44 translocations, suggesting that these translocations were the results of illegitimate switch recombination facilitated by homologous sequences present on both chromosomes. By interphase-FISH using home-grown CD44 dual-color break-apart probes (consisting of BACs RP4-607I7 and RP4-683L5), breakage at the CD44 locus in each of the nine IGHSμ/CD44 translocation-positive cases was confirmed. By 5’ RACE analysis, fusion Iμ-CD44ΔEx1 hybrid transcripts (with a splicing of the Iμ exon upstream of Sμ to exon 2 of CD44), were identified in all the nine lymphomas with IGHSμ/CD44 translocations. Notably, these translocations were detected exclusively in GCB-type DLBCLs. However, CD44 mRNA was minimally or not expressed in CD10+ microdissected reactive GCB cells. The IGHSμ/CD44 translocation substitute Sμ for the CD44 promoter and remove exon 1 of CD44, resulting in over-expression of the Iμ-CD44 hybrid transcript which encodes for a new CD44 variant lacking leader peptide sequence but retaining a unique C-terminus (CD44ΔEx1). The Iμ-CD44ΔEx1 ORF would encode for the CD44ΔEx1 protein starting from the ATG at nucleotide 254 with strong Kozak sequence. The new CD44ΔEx1 variant showed some similarity to CD44v5, but it lacked the leader peptide. The IGHSμ/CD44 translocation was detected in patients with advanced-stage disease (stages III and IV). When overexpressed in vitro in the CD44-negative GCB-DLBCL cell line BJAB, the CD44ΔEx1-GFP was localized to the cytoplasm and nucleus, while CD44s-GFP (standard form) was localized to the plasma membrane. The ectopic expression of CD44ΔEx1 in BJAB cells enhanced the cell proliferation rate and its clonogenic ability. These findings indicate a possible pathogenic role of the recurrent translocation in several malignant B-cell lymphomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 258.

Abstract LB-113: Non-small cell lung cancer cells with high ALDH1 activity display cancer stem cell properties and show chemoresistance

Introduction Cancer stem cells (CSC) are believed to possess tissue stem cell-like properties such as self-renewal and toxicity resistance. These characteristics would facilitate tumor propagation and development of chemoresistance. The biology and regulation of CSC have mostly been studied in acute leukemia and certain solid tumors such as breast cancer. Relatively little data are available regarding CSC in non-small cell lung cancer (NSCLC) due to the lack of a specific CSC marker. This study aims to analyze the applicability of candidate CSC markers by utilizing chemotherapy treatment to enhance CSC proliferation in NSCLC cells. Methodology The expression or activity of 4 putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase (ALDH1) were measured by flow cytometry in the NSCLC cell lines H1650, HCC827 and H358 before and after chemotherapy treatment for 24 hours. Tumor cells with enhanced marker expression after treatment were regarded as potential CSC, and were selected by fluorescence-activated cell sorting (FACS). Functional analysis was used to test for CSC properties in the marker+ and marker-subpopulations of untreated cells. The expression of genes involved in tissue stem cell functions were also compared by quantitative RT-PCR. Results Flow cytometry analysis showed amongst the 4 markers, only ALDH1+ subpopulation was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly higher numbers and larger cell spheres in non-adherent culture medium than ALDH1− cells. Likewise, ALDH1+ cells formed significantly more and larger colonies in colony formation assay. Furthermore, MTT assay demonstrated higher resistance to cisplatin and etoposide treatments in ALDH1+ than ALDH1− cells. In addition, ALDH1+ cells showed more prominent tumorigenicity than ALDH1− cells in vivo; as few as 500 ALDH1+ cells formed xenograft tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH− cells. Finally, expression analysis of sorted cells revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. Conclusion NSCLC cells expressing ALDH1 displayed higher capacity for cell renewal, tumorigenecity and drug resistance, as well as showed higher expression of pluripotency genes, suggesting that ALDH1 could be a useful CSC marker in NSCLC. Pathways mediated by the differentially expressed genes studied could be involved in CSC maintenance and/or chemoresistance in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2011-LB-113