Hun Soon Jung

Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications

Unlike epigenetic silencing of tumor suppressor genes, the role of epigenetic derepression of cancer-promoting genes or oncogenes in carcinogenesis remains less well understood. The tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer and their overexpression was previously reported to promote the migration and invasion of ovarian epithelial cells. Here, we show that the expression of claudin-3 and claudin-4 is repressed in ovarian epithelial cells in association with promoter ‘bivalent’ histone modifications, containing both the activating trimethylated histone H3 lysine 4 (H3K4me3) mark and the repressive mark of trimethylated histone H3 lysine 27 (H3K27me3). During ovarian tumorigenesis, derepression of CLDN3 and CLDN4 expression correlates with loss of H3K27me3 in addition to trimethylated histone H4 lysine 20 (H4K20me3), another repressive histone modification. Although CLDN4 repression was accompanied by both DNA hypermethylation and repressive histone modifications, DNA methylation was not required for CLDN3 repression in immortalized ovarian epithelial cells. Moreover, activation of both CLDN3 and CLDN4 in ovarian cancer cells was associated with simultaneous changes in multiple histone modifications, whereas H3K27me3 loss alone was insufficient for their derepression. CLDN4 repression was robustly reversed by combined treatment targeting both DNA demethylation and histone acetylation. Our study strongly suggests that in addition to the well-known chromatin-associated silencing of tumor suppressor genes, epigenetic derepression by the conversely related loss of repressive chromatin modifications also contributes to ovarian tumorigenesis via activation of cancer-promoting genes or candidate oncogenes.

Enhanced expression of hedgehog signaling molecules in squamous cell carcinoma of uterine cervix and its precursor lesions

The hedgehog (Hh)-signaling pathway plays an essential role in normal development. Deregulation of this pathway is responsible for several types of cancers. The aim of this study was to determine the expression pattern and the extent of Hh-signaling molecules in squamous cell carcinoma of uterine cervix and its precursor lesions. A total of 106 uterine cervical cancers and related lesions (37 squamous cell carcinomas, 23 cervical intraepithelial neoplasia (CIN) III, 10 CIN II, four CIN I, 32 normal cervical epithelia) were immunohistochemically analyzed with anti-Shh, Indian Hh (Ihh), Patched (PTCH), Smoothened (Smo), Gli-1, Gli-2, Gli-3 antibodies on paraffin blocks. The results showed that the expression of all the Hh-signaling molecules was greatly enhanced in uterine cervical tumors, including carcinoma and its precursor lesions. The staining pattern was mainly cytoplasmic except for Gli-1/2, whose expression was observed in both cytoplasm and nucleus. In case of Ihh, PTCH, Smo and Gli-1, their expression in normal epithelium was completely absent or rare. The expression of all the seven Hh-signaling molecules mentioned above was significantly increased in CIN II/III and carcinoma, compared with that in normal epithelium (P<0.05). The expression of Shh was increased by double; the first increase occurred in normal epithelium–CIN transition, and the second, during the progression of CIN to carcinoma. These results strongly suggest that the Hh-signaling pathways were extensively activated in carcinoma and CIN of uterine cervix. In conclusion, the Hh-signaling pathways may be involved in carcinogenesis of squamous cell carcinoma of uterine cervix and can be considered as a potential therapeutic target.

Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma

The tight junction (TJ) protein claudin-4 is aberrantly upregulated in gastric cancer, but its clinical significance and the molecular mechanisms underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanisms regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression did not show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it did not affect cell growth. Claudin-4 expression also increased the barrier function of TJs. Claudin-4 upregulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, CLDN4 expression was repressed in normal gastric tissues in association with bivalent histone modifications, and loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression and that it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer.

The synergistic therapeutic effect of cisplatin with Human papillomavirus E6/E7 short interfering RNA on cervical cancer cell lines in vitro and in vivo

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis‐diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7‐specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7‐specific siRNA had greater efficacy than the combination of CDDP and E6‐specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7‐specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long‐term exposure of HeLa cells to the combination of CDDP and E6/E7‐specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7‐specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7‐specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.

Human Papillomavirus: Current and Future RNAi Therapeutic Strategies for Cervical Cancer

Human papillomaviruses (HPVs) are small DNA viruses; some oncogenic ones can cause different types of cancer, in particular cervical cancer. HPV-associated carcinogenesis provides a classical model system for RNA interference (RNAi) based cancer therapies, because the viral oncogenes E6 and E7 that cause cervical cancer are expressed only in cancerous cells. Previous studies on the development of therapeutic RNAi facilitated the advancement of therapeutic siRNAs and demonstrated its versatility by siRNA-mediated depletion of single or multiple cellular/viral targets. Sequence-specific gene silencing using RNAi shows promise as a novel therapeutic approach for the treatment of a variety of diseases that currently lack effective treatments. However, siRNA-based targeting requires further validation of its efficacy in vitro and in vivo, for its potential off-target effects, and of the design of conventional therapies to be used in combination with siRNAs and their drug delivery vehicles. In this review we discuss what is currently known about HPV-associated carcinogenesis and the potential for combining siRNA with other treatment strategies for the development of future therapies. Finally, we present our assessment of the most promising path to the development of RNAi therapeutic strategies for clinical settings.

Merkel cell carcinoma: Our experience with seven patients in Korea and a literature review

BACKGROUND Merkel cell carcinoma (MCC) is a rare but malignant cutaneous neuroendocrine carcinoma. As MCC has primarily been reported in Caucasians, MCC cases in Korea have not yet been reported. The purpose of this study was to retrospectively review our experience with the surgical treatment of MCC in Korea and to study its management and outcome. METHOD We retrospectively reviewed seven MCC case files between 2000 and 2008 from a single institution. We analysed patient characteristics, tumour location and size, staging, treatment methods and outcomes. We performed polymerase chain reaction (PCR) to detect Merkel cell polyomavirus (MCPyV) from formalin-fixed paraffin-embedded tissue specimens. RESULTS Two patients had stage I tumours, four patients had stage II tumours and one patient had a stage III tumour. Wide local excision with a clear resection margin was the primary modality of treatment in all cases. Adjuvant radiotherapy and chemotherapy were performed for selected patients. Recurrence was observed in two out of the seven cases during the follow-up period. MCPyV was detected by PCR in all seven cases. CONCLUSION MCC is an aggressive skin cancer, and pathologic lymph node evaluation is important for staging. Wide excision is the primary modality of treatment, but adjuvant radiotherapy could be positively considered if the tumour is large and the lesion is not confined to the dermis. MCPyV was detected by PCR in all cases, which suggests that MCPyV is also a putative aetiological agent in the carcinogenesis of MCC in Korea.

Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo

The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

Low SP1 Expression Differentially Affects Intestinal-Type Compared with Diffuse-Type Gastric Adenocarcinoma

Specificity protein 1 (SP1) is an essential transcription factor that regulates multiple cancer-related genes. Because aberrant expression of SP1 is related to cancer development and progression, we focused on SP1 expression in gastric carcinoma and its correlation with disease outcomes. Although patient survival decreased as SP1 expression increased (P<0.05) in diffuse-type gastric cancer, the lack of SP1 expression in intestinal-type gastric cancer was significantly correlated with poor survival (P<0.05). The knockdown of SP1 in a high SP1-expressing intestinal-type gastric cell line, MKN28, increased migration and invasion but decreased proliferation. Microarray data in SP1 siRNA-transfected MKN28 revealed that the genes inhibiting migration were downregulated, whereas the genes negatively facilitating proliferation were increased. However, both migration and invasion were decreased by forced SP1 expression in a low SP1-expressing intestinal-type gastric cell line, AGS. Unlike the intestinal-type, in a high SP1-expressing diffuse-type gastric cell line, SNU484, migration and invasion were decreased by SP1 siRNA. In contrast to previous studies that did not identify differences between the 2 histological types, our results reveal that low expression of SP1 is involved in cancer progression and metastasis and differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.

MET Exon 14 Skipping Mutations in Lung Adenocarcinoma: Clinicopathologic Implications and Prognostic Values

Introduction Response to mesenchymal‐epithelial transition (MET) inhibitors in NSCLC with mesenchymal‐epithelial transition gene (MET) exon 14 skipping (METex14) has fueled molecular screening efforts and the search for optimal therapies. However, further work is needed to refine the clinicopathologic and prognostic implications of METex14 skipping. Methods Among 795 East Asian patients who underwent a surgical procedure for NSCLC, we screened 45 patients with quintuple‐negative (EGFR‐negative/KRAS‐negative/anaplastic lymphoma kinase gene [ALK]‐negative/ROS1‐negative/ret proto‐oncogene [RET]‐negative) lung adenocarcinomas by using reverse‐transcriptase polymerase chain reaction and found 17 patients (37.8%) with METex14 skipping. We also investigated the effect of small interfering RNA (siRNA) targeting skipping junction in cells with METex14 skipping. Results The median age of the 17 patients was 73 years. The acinar subtype was predominant (52.9%), followed by the solid subtype (35.3%). MET immunohistochemistry demonstrated 100% sensitivity and 70.4% specificity. Multivariate analyses showed that patients with METex14 skipping had a higher recurrence rate than those with ALK fusion (versus METex14 skipping) (hazard ratio = 0.283, 95% confidence interval: 0.119–0.670) in stage I to IIIA disease; however, the differences in overall survival were not significant after adjustment for pathologic stage (p = 0.669). Meanwhile, siRNA decreased MET‐driven signaling pathways in Hs746T cells, and combined treatment with siRNA and crizotinib inhibited cell proliferation in crizotinib‐resistant H596 cells. Conclusions The prevalence of METex14 skipping was quite high in East Asian patients without other driver mutations in lung adenocarcinomas. METex14 skipping was associated with old age, the acinar or solid histologic subtype, and high MET immunohistochemical expression. The prognosis of patients with METex14 skipping was similar to that of patients with major driver mutations. siRNA targeting the junction of METex14 skipping could inhibit MET‐driven signaling pathways in cells with METex14 skipping.

The potential RNAi-based combination therapeutics

Recently, antiviral RNA interference (RNAi) usingshort-interfering RNA (siRNA) has been developed asa novel strategy and tested in clinical trials. siRNAshave been demonstrated to selectively silence not onlyendogenous genes in mammalian cells (Sui et al.,2002; Yu et al., 2002) but also viral genes in virus-induced diseases (Kitabwalla and Ruprecht, 2002; Geet al., 2003; Milner, 2003). Several RNAi studies wereperformed with human tumor cell lines using syn-thetic siRNAs or short hairpin RNAs (shRNAs) li-braries to identify the modulators of anticancer drugsensitivity (Bartz et al., 2006; Ji et al., 2007; Swantonet al., 2007; Whitehurst et al., 2007; Honma et al.,2008; Bauer et al., 2010). Combination therapy withsiRNA may significantly enhance the sensitivity ofcancer cells to the anticancer therapy and thereby helpprevent the development of Chemo/Radio resistanceresulting from low-dose Chemo/Radio therapy (Fig. 1).Previously, the approach using synthesized siRNApools was employed to identify the chemosensitizer inNCI-H1155, a human non-small-cell lung cancer line(Whitehurst et al., 2007). A set of 87 candidate pacli-taxel-sensitizing genes, including receptor–ligand pairs,proteasome, microtubules, testis antigens, and Rasfamily, were identified. In addition, a major contribu-tion of mitotic progression-related genes in cancercells was also highlighted. Other study performed byJung validated the role of 2-in-1 antiviral siRNAtargeting human papillomavirus (HPV)