Sook Moon

Human telomerase reverse transcriptase (hTERT) promotes cancer invasion by modulating cathepsin D via early growth response (EGR)-1

Human telomerase reverse transcriptase (hTERT) contributes to tumor progression as well as maintaining telomere length, however, the mechanism by which hTERT promotes invasiveness is not yet completely understood. This study aims to unravel the precise mechanism through which hTERT promotes cancer invasion. We established an hTERT-overexpressed immortalized cell line (IHOK/hTERT). In orthotopic xenograft models, IHOK/hTERT harbors higher tumorigenicity than IHOK/Control. IHOK/hTERT showed much higher migration and invasion activities compared to IHOK/Control. IHOK/hTERT co-cultured with fibroblasts displayed increased invasion compared to IHOK/hTERT without fibroblasts. We screened for genes that play an important role in intermodulation between cancer cells and fibroblasts using a microarray and identified fibroblast activation protein (FAP). hTERT knockdown showed decreased expression of FAP and early growth response (EGR)-1, one of the transcriptional regulators of FAP in IHOK/hTERT and oral cancer cell line YD10B. Furthermore, EGR-1 knockdown in IHOK/hTERT and YD10B showed reduced invasion and reduced cathepsin D expression compared to Control-siRNA cells. Taken together, this study provides evidence that hTERT overexpression is responsible for the upregulation of the cysteine protease cathepsin D by regulating EGR-1 to activate invasiveness in cancer progression.

Apoptosis-related microRNA-145-5p enhances the effects of pheophorbide a-based photodynamic therapy in oral cancer

MicroRNAs (miRNAs) regulate key biological processes, and their aberrant expression has been related to cancer development. Photodynamic therapy (PDT) has emerged as one of the most promising modalities for cancer treatment. However, the application of PDT has been limited to superficially localized human cancerous and precancerous lesions. To increase the usefulness of both PDT and miRNAs in cancer therapy, this study investigated whether apoptosis-related miRNA expression is influenced by PDT in oral cancer and whether miRNAs can enhance PDT efficacy. To achieve this goal, we performed a miRNA array-based comparison of apoptosis-related miRNA expression patterns following PDT using pheophorbide a (Pa) as a photosensitizer. After Pa-PDT, 13.1% of the miRNAs were down-regulated, and 16.7% of the miRNAs were up-regulated. Representative miRNAs were selected according to expression difference: miR-9-5p, miR-32-5p, miR-143-3p, miR-145-5p, miR-192-5p, miR-193a-5p, miR-204-5p, miR-212-3p, miR-338-3p, and miR-451a. Among them, only miR-145-5p showed the consistent reduction repeatedly in all cell lines after Pa-PDT. Further, the combined treatment of a miR-145-5p mimic and Pa-PDT increased phototoxicity, reactive oxygen species generation, and apoptotic cell death, suggesting that miRNAs expression could be a useful marker for enhancing the therapeutic effect of Pa-PDT. This study will provide a promising strategy for introducing miRNA as cancer therapy.

RUNX3 expression is associated with sensitivity to pheophorbide a-based photodynamic therapy in keloids

Runt-related transcription factor 3 (RUNX3) has recently been reported to be a possible predictor of sensitivity of cancer cells for photodynamic therapy (PDT), a promising therapeutic modality for keloids. In this study, we aimed to elucidate the implications of RUNX3 for keloid pathogenesis and sensitivity to pheophorbide a-based PDT (Pa-PDT). RUNX3 and proliferating cell nuclear antigen (PCNA) expression were examined in 6 normal skin samples and 32 keloid tissue samples by immunohistochemistry. We found that RUNX3 expression was detected more often in keloid tissues than in dermis of normal skin. In keloid tissues, RUNX3 expression was significantly increased in patients presenting with symptoms of pain or pruritus, and was also significantly related to PCNA expression. The therapeutic effect of Pa-PDT was comparatively investigated in keloid fibroblasts (KFs) with and without RUNX3 expression. Significant differences were found after Pa-PDT between KFs with and without RUNX3 expression in cell viability, proliferative ability, type I collagen expression, generation of reactive oxygen species (ROS), and apoptotic cell death. In addition, RUNX3 expression was significantly decreased after Pa-PDT in KFs, and KFs with downregulation of RUNX3 showed significantly increased cell viability after Pa-PDT. Pa-PDT may be a potential therapeutic modality for keloids, and RUNX3, as a possible contributor to keloid pathogenesis, may improve sensitivity to Pa-PDT in KFs.

CXCL1 induces senescence of cancer-associated fibroblasts via autocrine loops in oral squamous cell carcinoma

Cancer-associated fibroblasts (CAFs) have emerged as one of the main factors related to cancer progression, however, the conversion mechanism of normal fibroblasts (NOFs) to CAFs has not been well elucidated. The aim of this study was to investigate the underlying mechanism of CAF transformation from NOFs in oral squamous cell carcinoma (OSCC). This study found that NOFs exposed to OSCC cells transformed to senescent cells. The cytokine antibody array showed the highest secretion levels of IL-6 and CXCL1 in NOFs co-cultured with OSCC cells. Despite that both IL-6 and CXCL1 induced the senescent phenotype of CAFs, CXCL1 secretion showed a cancer-specific response to transform NOFs into CAFs in OSCC, whereas IL-6 secretion was eventuated by common co-culture condition. Further, CXCL1 was released from NOFs co-cultured with OSCC cells, however, CXCL1 was undetectable in mono-cultured NOFs or co-cultured OSCC cells with NOFs. Taken together, this study demonstrates that CXCL1 can transform NOFs into senescent CAFs via an autocrine mechanism. These data might contribute to further understanding of CAFs and to development of a potential therapeutic approach targeting cancer cells-CAFs interactions.

Insulin-like growth factor-II mRNA binding protein-3 and podoplanin expression are associated with bone invasion and prognosis in oral squamous cell carcinoma

OBJECTIVE This study aimed to evaluate the prognostic implications of insulin-like growth factor-II mRNA binding protein-3 (IMP3) and podoplanin (PDPN) as therapeutic targets against oral squamous cell carcinoma (OSCC) with bone invasion. STUDY DESIGN We elucidated the correlation of IMP3 and PDPN expression with bone invasion in 160 OSCC tissue specimens, and assessed a mouse calvarium xenograft model using an IMP3- and PDPN-depleted OSCC cell line. RESULTS The retrospective analysis revealed that the expression of IMP3 and PDPN is significantly correlated with T stage, lymph node metastasis, and the overall survival of OSCC patients. In addition, the dual expression of IMP3 and PDPN but not the single expression of either IMP3 or PDPN was associated with bone invasion and the number of osteoclasts in patients with OSCC. In support of these findings, IMP3 or PDPN depletion inhibited the invasive capacity of OSCC cells in a three-dimensional culture system, tumorigenesis, and regional bone destruction in a xenograft mouse model. In addition, IMP3 or PDPN depletion inhibited the expression of interleukin (IL)-6 and IL-8 in OSCC cells, and decreased the expression of receptor activator of NF-κB ligand (RANKL) in xenograft tumor tissues of OSCC. CONCLUSIONS These results suggest that IMP3 and PDPN may have strong influence on the pathogenesis of OSCC, especially in bone invasion, and may serve as novel therapeutic targets with prognostic implications for bone-invasive OSCC.

Abstract 176: IL6 and CXCL1 induce senescent phenotype of cancer-associated fibroblast via autocrine loops in oral squamous cell carcinoma

Background & Objective Tumor-stroma interaction plays a key role in tumor development and progression. One of the prominent components of tumor stroma is cancer-associated fibroblasts (CAFs). Although CAFs have been well known to contribute to tumorigenesis, the characteristics of CAFs are still poorly understood. For this reason, we characterized CAFs and investigated a mechanism underlying the transformation of NOFs into CAFs in oral squamous cell carcinoma (OSCC). Materials & Methods For this study, three primary cultured NOFs and three primary cultured CAFs were used, respectively. Expression of proliferating cell nuclear antigen (PCNA) and senescence-associated β-galactosidase (SA-β-Gal) enzyme activity were compared between NOFs and CAFs. Population doubling levels and expression of activated fibroblast marker were measured in NOFs and CAFs. The expression levels of senescence-related markers were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. In search of mechanism that triggers senescence in CAFs, cytokine antibody array was employed in NOFs co-cultured with OSCC cells, and the results were confirmed by real-time PCR. Results In comparison between NOFs and CAFs, α-SMA, a marker of activated fibroblasts, showed no difference in expression pattern. CAFs showed higher SA-β-Gal enzyme activity and lower PCNA expression than NOFs at the same passage. In addition, CAFs exhibited lower population doubling level than NOFs, indicating that CAFs had senescent phenotype. NOFs co-cultured with OSCC cells showed higher SA-β-Gal enzyme activity, p16 and p21 expression compared with mono-cultured NOFs, whereas NOFs co-cultured with human epidermal keratinocytes (HEK) showed no SA-β-Gal enzyme activity, indicating that the induction of senescence in CAFs was not merely an artifact of co-culture system but was triggered specifically by the co-cultured cancer cells. Cytokine antibody array revealed that co-culture conditions induced cytokine secretion from CAFs. In particular, IL6 and CXCL1 showed the highest secretion level, and mRNA expression levels corresponded with the results from cytokine antibody array. Upon treating NOFs with IL6 and CXCL1, higher SA-β-Gal enzyme activity was detected in NOFs compared with non-treated NOFs, indicating that IL6 and CXCL1 were capable of inducing senescence in NOFs. Conclusion From these results, we propose that the senescent phenotype of CAFs might be elicited by cytokines such as IL6 and CXCL1, which are secreted from CAFs in an autocrine manner. Additional studies are in progress to identify specific factors to induce cytokine secretion in a carcinoma milieu. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2009-0094027). Citation Format: Eun Kyoung Kim, Sook Moon, Do Kyeong Kim, Jin Kim, Jung Yoon Bae. IL6 and CXCL1 induce senescent phenotype of cancer-associated fibroblast via autocrine loops in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 176. doi:10.1158/1538-7445.AM2014-176

Abstract 3549: RUNX3 is a biomarker for determining sensitivity to pheophorbide a-photodynamic therapy in human oral squamous cell carcinoma.

Background & Objective: Photodynamic therapy (PDT) with photosensitizer is one of the promising modalities for cancer treatment. For clinical use of PDT, the screening process for sensitive cancers to PDT should be preceded. For this, we investigated a molecular biomarker to determine the sensitivity to Pa-PDT in immortalized human oral keratinocytes (IHOK) and OSCC cell lines. Methods: For this study, two immortalized oral keratinocytes (IHOK-p, IHOK-s) and 8 oral squamous cell carcinoma (OSCC) cell lines were used. After cells were treated with Pa-PDT, phototoxicity and the level of reactive oxygen species (ROS) were measured. Apoptosis was measured using annexin V/PI staining and western blotting. mRNA and proteins of apoptotic genes were investigated by RT-PCR, real-time PCR, and western blotting. Transfection was performed using RUNX3-small interfering RNA. Results: After Pa-PDT, cell viability was more than 50% reduced and ROS generated in IHOK and OSCC. In addition, apoptosis occurred in all cell lines. Among apoptosis-related genes, Bim expression was altered following Pa-PDT. Therefore, mRNA and protein expression of RUNX3, a gene upstream of Bim were examined by Pa-PDT. We found that RUNX3 was highly responsive to Pa-PDT. Furthermore, knockdown of RUNX3 expression reduced cytotoxicity by Pa-PDT. In addition, we found that the cytotoxicity by Pa-PDT was proportional to RUNX3 expression in OSCC cell lines. Conclusion: This is the first study to find a new target molecule that enhances Pa-PDT effects in IHOK and OSCC cell lines. These results should be further proven by animal studies to apply to clinical trials. Nevertheless, the development of a PDT-dependent biomarker could provide a novel approach to improve the effects of PDT in oral precancerous and cancerous lesions. Acknowledgments This study was supported by a grant of the Korean Health Technology RD 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3549. doi:10.1158/1538-7445.AM2013-3549

Abstract B74: EGCG inhibits cancer invasion by regulating tumor-stromal crosstalk in oral squamous cell carcinoma

Introduction : Epigallocatechin-3-O-gallate (EGCG), a major constituent polyphenol of green tea, has been shown to have suppressive effects on the invasion of various cancer cells, whereas the exact mechanisms have not yet been fully elucidated. In this study, the effects of EGCG on the invasion of oral squamous cell carcinoma (OSCC) were investigated where the new perspective focused on tumor-stromal crosstalk was emphasized, unlikely other previous studies focused on cancer cell mainly. Materials and Methods : OSCC were cultured with or without carcinoma-associated fibroblasts (CAF) in the dose dependent treatment of EGCG. In order to investigate the invasiveness, invasion assay and zymography were examined. Based on our previous study found that several cytokines are concerned with tumor-stromal crosstalk in invasion of OSCC, RT-PCR and ELISA assay were employed to examine the influence of EGCG on the expression levels of cytokines. Results : The invasiveness of OSCC was reduced by EGCG treatment, and the 50% reduction was demonstrated at the half concentration of EGCG (25 μM) in co-cultured group with CAF, compared to OSCC mono-cultured (50 μM). Zymography showed that EGCG treatment reduced MMP-9 expression in co-cultured condition. In addition, EGCG resulted in a noticeable decrease in the expression of cytokines, especially GRO-α/CXCL1, concerned with tumor-stromal crosstalk in invasion of OSCC. Conclusions : In this study, inhibitory effect of EGCG on cancer invasion was found at which its concentration is lower than previously known. Taking these results into consideration, EGCG may play an effective role in preventing invasion of OSCC by regulating tumor-stromal crosstalk. Acknowledgement : This work was supported by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010–0029702). Citation Information: Cancer Prev Res 2011;4(10 Suppl):B74.