Jung Yoon Bae

Reciprocal interaction between carcinoma-associated fibroblasts and squamous carcinoma cells through interleukin-1α induces cancer progression.

Crosstalk between cancer cells and carcinoma-associated fibroblasts (CAFs) has earned recognition as an interaction that plays a pivotal role in carcinogenesis. Thus, we attempted to clarify whether increase in the level of CAFs promotes cancer progression by proportionally enhancing the interaction between cancer cells and CAFs. We first analyzed clinical correlation between the levels of fibroblasts and cancer progression and found that the level of CAFs made a noticeable difference on the prognosis of patients with oral squamous cell carcinoma (OSCC). In vivo animal study also demonstrated that tumor volume depended on the dose of CAFs that was co-injected with OSCC cells. The same tendency was observed in an in vitro study. We also found that interleukin-1α (IL-1α) secreted from OSCC cells had dual effects on CAFs: IL-1α not only promoted the proliferation of CAFs but also upregulated the secretion of cytokines in CAFs such as CCL7, CXCL1, and IL-8. The induction activity of cytokine secretion by IL-1α surpassed that of proliferation in OSCC cells. In summary, we unraveled an important interactive mechanism of carcinogenesis: IL-1α released from carcinoma stimulates the proliferation of CAFs and the simultaneous increase in cytokine secretion from CAFs promotes cancer progression in human OSCC. On the basis of these findings, we propose that the level of CAFs is eligible for being selected as a prognostic factor that will be useful in routine diagnosis. We also propose that blockage of reciprocal interaction between cancer cells and CAFs will provide an insight for developing novel chemotherapeutic strategy.

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.

A distinct role for interleukin-6 as a major mediator of cellular adjustment to an altered culture condition.

Tissue microenvironment adjusts biological properties of different cells by modulating signaling pathways and cell to cell interactions. This study showed that epithelial–mesenchymal transition (EMT)/ mesenchymal–epithelial transition (MET) can be modulated by altering culture conditions. HPV E6/E7‐transfected immortalized oral keratinocytes (IHOK) cultured in different media displayed reversible EMT/MET accompanied by changes in cell phenotype, proliferation, gene expression at transcriptional, and translational level, and migratory and invasive activities. Cholera toxin, a major supplement to culture medium, was responsible for inducing the morphological and biological changes of IHOK. Cholera toxin per se induced EMT by triggering the secretion of interleukin 6 (IL‐6) from IHOK. We found IL‐6 to be a central molecule that modulates the reversibility of EMT based not only on the mRNA level but also on the level of secretion. Taken together, our results demonstrate that IL‐6, a cytokine whose transcription is activated by alterations in culture conditions, is a key molecule for regulating reversible EMT/MET. This study will contribute to understand one way of cellular adjustment for surviving in unfamiliar conditions. J. Cell. Biochem. 116: 2552–2562, 2015.

Abstract 3354: The level of aerobic glycolysis as an effective predictor of tumorigenicity

Introduction: Cancer cells have been known to harbor characteristic metabolic alterations such as enhanced glucose uptake and aerobic glycolysis. However, elucidation of molecular mechanisms underlying the association between aerobic glycolysis and tumorigenesis has remained elusive. Thus, this study aimed to investigate the role of altered metabolism in conferring advantage to tumorigenesis, using two different cell lines of immortalized human oral keratinocytes (IHOK) that differed in the level of aerobic glycolysis. Experimental procedures: Levels of glucose uptake and lactate production were measured. Reverse transcription polymerase chain reaction and western blot were employed to measure the expression of relevant genes. Proteomic analysis was performed to identify protein-level differences. Small interfering RNAs were used for transient knockdown of genes. The cell lines were injected into mouse tongue to assess subsequent tumor formation. Results: Although the two IHOK cell lines shared same origin, the cell line with enhanced glucose uptake and lactate production also had higher expression of genes involved in glycolysis, growth signal propagation, and matrix remodeling. In particular, matrix metalloproteinase (MMP)-1, 2, 9, and 14 expressions were significantly higher in the cell line with upregulated glycolysis. Upon injection into mice, the two cell lines differed in the rate of in vivo tumor formation and resulting tumor volume. Epidermal growth factor receptor (EGFR) was upregulated in the more tumorigenic IHOK cell line as well as in aggressive breast and oral cancer cell lines compared with their less aggressive counterparts. EGFR knockdown in the more tumorigenic cell lines led to reduction in lactate production and prevented high-level expression of glycolytic genes normally induced by EGF treatment. Proteomic analysis between the two IHOK cell lines revealed pyruvate kinase isozyme M2 (PKM2) as the protein whose level was significantly higher in the more tumorigenic cell line. To evaluate the relationship between upregulated PKM2 and high tumorigenic potentials, PKM2 was knocked down in the more tumorigenic cell line. MMP-1 level decreased upon PKM2 knockdown, indicating that upregulated PKM2 not only promotes aerobic glycolysis but also enhances MMP expression to facilitate tumorigenesis. Conclusions: Cells with upregulated PKM2 likely excel in tumorigenesis by enhancing MMP expression. In addition, upregulated EGFR amplifies the growth signals to enhance the expression of genes required for tumor progression. These results indicate that the degree of altered metabolic activity, aerobic glycolysis in particular, might be a significant predictor of tumorigenicity. Acknowledgements: 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: Doo Young Lee, Young Jin Park, Jung Yoon Bae, Jin Kim. The level of aerobic glycolysis as an effective predictor of tumorigenicity. [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 3354. doi:10.1158/1538-7445.AM2014-3354

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