Wen Son Hsieh

The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2′-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkins lymphoma, 3 of 6 (50%) Hodgkins lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2′-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

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.

Pharmacodynamic Effects of Seliciclib, an Orally Administered Cell Cycle Modulator, in Undifferentiated Nasopharyngeal Cancer

Purpose: Cell cycle dysregulation resulting in expression of antiapoptotic genes and uncontrolled proliferation is a feature of undifferentiated nasopharyngeal carcinoma. The pharmacodynamic effects of seliciclib, a cyclin-dependent kinase (CDK) inhibitor, were studied in patients with nasopharyngeal carcinoma. Experimental Design: Patients with treatment-naïve locally advanced nasopharyngeal carcinoma received seliciclib at 800 mg or 400 mg twice daily on days 1 to 3 and 8 to 12. Paired tumor samples obtained at baseline and on day 13 were assessed by light microscopy, immunohistochemistry, and transcriptional profiling using real-time PCR low-density array consisting of a panel of 380 genes related to cell cycle inhibition, apoptosis, signal transduction, and cell proliferation. Results: At 800 mg bd, one patient experienced grade 3 liver toxicity and another had grade 2 vomiting; no significant toxicities were experienced in 13 patients treated at 400 mg bd. Seven of fourteen evaluable patients had clinical evidence of tumor reduction. Some of these responses were associated with increased tumor apoptosis, necrosis, and decreases in plasma EBV DNA posttreatment. Reduced protein expression of Mcl-1, cyclin D1, phosphorylated retinoblastoma protein pRB (T821), and significant transcriptional down-regulation of genes related to cellular proliferation and survival were shown in some patients posttreatment, indicative of cell cycle modulation by seliciclib, more specifically inhibition of cdk2/cyclin E, cdk7/cyclin H, and cdk9/cyclin T. Conclusions: Brief treatment with this regimen of seliciclib in patients with nasopharyngeal carcinoma is tolerable at 400 mg bd and associated with tumor pharmacodynamic changes consistent with cdk inhibition, and warrants further efficacy studies in this tumor.

A novel isoform of the 8p22 tumor suppressor gene DLC1 suppresses tumor growth and is frequently silenced in multiple common tumors

The critical 8p22 tumor suppressor deleted in liver cancer 1 (DLC1) is frequently inactivated by aberrant CpG methylation and/or genetic deletion and implicated in tumorigeneses of multiple tumor types. Here, we report the identification and characterization of its new isoform, DLC1 isoform 4 (DLC1-i4). This novel isoform encodes an 1125-aa (amino acid) protein with distinct N-terminus as compared with other known DLC1 isoforms. Similar to other isoforms, DLC1-i4 is expressed ubiquitously in normal tissues and immortalized normal epithelial cells, suggesting a role as a major DLC1 transcript. However, differential expression of the four DLC1 isoforms is found in tumor cell lines: Isoform 1 (longest) and 3 (short thus probably nonfunctional) share a promoter and are silenced in almost all cancer and immortalized cell lines, whereas isoform 2 and 4 utilize different promoters and are frequently downregulated. DLC1-i4 is significantly downregulated in multiple carcinoma cell lines, including 2/4 nasopharyngeal, 8/16 (50%) esophageal, 4/16 (25%) gastric, 6/9 (67%) breast, 3/4 colorectal, 4/4 cervical and 2/8(25%) lung carcinoma cell lines. The functional DLC1-i4 promoter is within a CpG island and is activated by wild-type p53. CpG methylation of the DLC1-i4 promoter is associated with its silencing in tumor cells and was detected in 38–100% of multiple primary tumors. Treatment with 5-aza-2′-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B led to demethylation of the promoter and reactivation of its expression, indicating a predominantly epigenetic mechanism of silencing. Ectopic expression of DLC1-i4 in silenced tumor cells strongly inhibited their growth and colony formation. Thus, we identified a new isoform of DLC1 with tumor suppressive function. The differential expression of various DLC1 isoforms suggests interplay in modulating the complex activities of DLC1 during carcinogenesis.

Consensus recommendations for management of head and neck cancer in Asian countries: A review of international guidelines

Head and neck cancer (HNC) is a disease of the upper aerodigestive tract and is one of the most frequently diagnosed cancers worldwide. A high rate of cancers involving the head and neck are reported across the Asian region, with notable variations between countries. Disease prognosis is largely dependent on tumor stage and site. Patients with early stage disease have a 60-95% chance of cure with local therapy. Early diagnosis and appropriate treatment are important to increase the likelihood of cure and survival. However, the majority of patients present with locally advanced disease and require multimodality treatment. This necessitates, a multidisciplinary approach which is essential to make appropriate treatment decisions, particularly with regards to tolerability, costs, available infrastructure and quality of life issues. Unfortunately, majority of the studies that dictate current practice have been developed in the west where diseases biology, patient population and available infrastructure are very different from those in the Asian continent. With this in mind an expert panel of Head and Neck Oncologists was convened in May 2012 to review the National Comprehensive Cancer Network (NCCN) and the European Society for Medical Oncology (ESMO) clinical practice guidelines and develop practical recommendations on the applicability of these guidelines on the management of head and neck cancer for Asian patients. The objective of this review and consensus meeting was to suggest revisions, to account for potential differences in demographics and resources, to the NCCN and ESMO guidelines, to better reflect current clinical management of head and neck cancer within the Asian region for health care providers. These recommendations, which reflect best clinical practice within Asia, are expected to benefit practitioners when making decisions regarding optimal treatment strategies for their patients.

A case of sweet’s syndrome due to 5-Azacytidine and vorinostat in a patient with NK/T cell lymphoma

Clinical trials are now evaluating hypomethylating agents in combination with histone deactylase inhibitors for nasopharyngeal carcinoma and NK/T cell lymphoma. (ClinicalTrials.gov: NCT00336063). When these drugs are used in myelodysplastic syndrome (MDS), there have been anecdotal reports of fulminant steroid refractory SS (1). Interestingly, all of these patients had an unusual histopathological feature of fewer invading neutrophils in their skin biopsy (1).

Abstract 3098: The effects of a pan-cyclin dependent kinase (CDK) inhibitor and its combination with cisplatin in nasopharyngeal carcinoma (NPC)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Undifferentiated NPC is a unique epithelial malignancy endemic to South-east Asia and North Africa. Cell cycle dysregulation, mediated by mechanisms such as over-expression of cyclin D1 and down-regulation of p16INK4A is frequently found in NPC. Our previous work suggest that NPC cells are sensitive to the effects of cell cycle inhibition. BAY1000394 (BAY) is a potent pan-CDK inhibitor exhibiting broad anti-proliferation activity across a number of different cancer types. In this study, the anti-tumor effects of BAY alone and in combination with cisplatin on NPC cell lines were examined in vitro (MTS assay) and in a xenograft model. Cell cycle effects and apoptosis were examined via flowcytometry. Protein levels of relevant cell cycle regulators and mediators of apoptosis were assessed via Western Blot. Time-dependent inhibition of NPC cell lines HONE-1 and HK-1 by BAY was evaluated and demonstrated that growth inhibition of 50% of the cell population occurred by 48hrs of treatment. Mean IC50 of 30nM was seen. BAY inhibits phosphorylation of cellular CDK target proteins such as retinoblastoma (Ser807/811) (Rb), nucleophosmin (Thr199) (NPM) and RNA polymerase II (Ser2) (p-RNA POL II). Dephosphorylation of RNA POL II inhibits transcription of gene products resulting in rapid decline of MCL-1. Direct caspase-3 activation was observed with the concomitant appearance of active caspase-3; inducing PARP cleavage and inhibition of survivin. BAY was able to induce cell cycle arrest at multiple checkpoints thus enhancing apoptosis. Annexin V and PI staining after 96hrs of BAY exposure showed apoptosis of >80% of BAY treated cell. In vivo dosing of BAY1000394 at 0.5mg/kg and 1mg/kg showed significant growth inhibition as compared to control (p-value <0.05). Combination studies of BAY with cisplatin displayed synergistic cell kill. Cell cycle analysis of HONE-1 cells treated at 10nM and 20nM BAY with doses of cisplatin ranging from 0.5μM to 2μM showed marked increase in cell death as compared to control and NP69 epithelial cells. Protein expression of activated caspase-3, PARP cleavage and MCL-1 inhibition were observed in dual drug treatment groups. No change in expressions these proteins were seen in treated NP69 cells. In vivo assays of both drugs also demonstrated growth inhibition of tumor bearing mice at BAY 0.5mg/kg and 1.0mg/kg with cisplatin 6mg/kg with good tolerance. Tumor protein expression of p-RNA POL II, NPM and MCL-1 expression were suppressed, suggesting inhibition of CDK9/cyclin T1. Our data suggest that BAY inhibits NPC tumor cell proliferation in a time- and dose- dependent manner through inhibition of cell-cycle progression and induction of apoptosis. Combination studies with cisplatin suggest synergistic anti-tumor effect. This preliminary study strongly suggests that BAY and its combination with cisplatin has therapeutic potential in the treatment of NPC. Citation Format: Pei Li Lim, John SW Low, Gerhard Siemeister, Boon Cher Goh, Wen-son Hsieh. The effects of a pan-cyclin dependent kinase (CDK) inhibitor and its combination with cisplatin in nasopharyngeal carcinoma (NPC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3098. doi:10.1158/1538-7445.AM2015-3098