You-Soub Lee

Runx3 Inactivation Is a Crucial Early Event in the Development of Lung Adenocarcinoma

Targeted inactivation of Runx3 in mouse lung induced mucinous and nonmucinous adenomas and markedly shortened latency of adenocarcinoma formation induced by oncogenic K-Ras. RUNX3 was frequently inactivated in K-RAS mutated human lung adenocarcinomas. A functional genetic screen of a fly mutant library and molecular analysis in cultured cell lines revealed that Runx3 forms a complex with BRD2 in a K-Ras-dependent manner in the early phase of the cell cycle; this complex induces expression of p14(ARF)/p19(Arf) and p21(WAF/CIP). When K-Ras was constitutively activated, the Runx3-BRD2 complex was stably maintained and expression of both p14(ARF) and p21(WAF/CIP) was prolonged. These results provide a missing link between oncogenic K-Ras and the p14(ARF)-p53 pathway, and may explain how cells defend against oncogenic K-Ras.

Runx3 is required for the differentiation of lung epithelial cells and suppression of lung cancer

Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molecular abnormalities. K-ras mutations are associated with the initiation of lung adenocarcinomas, but K-ras-independent mechanisms may also initiate lung tumors. Here, we find that the runt-related transcription factor Runx3 is essential for normal murine lung development and is a tumor suppressor that prevents lung adenocarcinoma. Runx3−/− mice, which die soon after birth, exhibit alveolar hyperplasia. Importantly, Runx3−/− bronchioli exhibit impaired differentiation, as evidenced by the accumulation of epithelial cells containing specific markers for both alveolar (that is SP-B) and bronchiolar (that is CC10) lineages. Runx3−/− epithelial cells also express Bmi1, which supports self-renewal of stem cells. Lung adenomas spontaneously develop in aging Runx3+/− mice (∼18 months after birth) and invariably exhibit reduced levels of Runx3. As K-ras mutations are very rare in these adenomas, Runx3+/− mice provide an animal model for lung tumorigenesis that recapitulates the preneoplastic stage of human lung adenocarcinoma development, which is independent of K-Ras mutation. We conclude that Runx3 is essential for lung epithelial cell differentiation, and that downregulation of Runx3 is causally linked to the preneoplastic stage of lung adenocarcinoma.

Jab1/CSN5 induces the cytoplasmic localization and degradation of RUNX3

Runt‐related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue‐specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun‐activation domain‐binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27Kip1, and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN‐associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome‐mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex. J. Cell. Biochem. 107: 557–565, 2009.

Sp1-associated activation of macrophage inflammatory protein-2 promoter by CpG-oligodeoxynucleotide and lipopolysaccharide.

Abstract.Macrophage inflammatory protein-2 (MIP-2) is a C-X-C chemokine that is important in recruiting neutrophils to inflammatory sites. Our previous reports demonstrated that lipopolysaccharide (LPS) or CpG-oligode-oxynucleotide (CpG-ODN) rapidly induce MIP-2 gene expression in the macrophage cell line, RAW 264.7. Here, we show that the DNA sequence of the MIP-2 promoter between −114 and +14 is sufficient for strong promoter activity in LPS- or CpG-ODN-stimulated RAW 264.7 cells. Importantly, comprehensive mutant analysis reveals that an Sp1 element in the promoter region between −114 and −94 is essential for synergistic MIP-2 promoter activation by NF-κB and c-Jun regardless of the presence of an AP-1 site. By combining deletion or site-specific mutant analysis with immunocomplex assays, we also confirmed that Sp1 mediates the recruitment of transcription factors NF- κB and c-Jun in LPS- or CpG-ODN-treated RAW 264.7 cells. Several lines of experimental evidence imply that the Sp1-binding element is an important determinant of MIP-2 promoter activity, and that NF-κB, c-Jun and Sp1 can functionally cooperate to elicit maximal activation of the promoter.

Runx3 is a crucial regulator of alveolar differentiation and lung tumorigenesis in mice.

The runt-domain transcription factor Runx3 plays crucial roles during development such as regulating gene expression. It has been shown that Runx3 is involved in neurogenesis, thymopoiesis and functions like a tumor suppressor. Runx3 null mouse die soon after birth as a result of multiple organ defects. Runx3 null mouse lung shows an abnormal phenotype and loss of Runx3 induced remodeling in the lung. Interestingly, lung adenocarcinoma is observed in Runx3 heterozygous mice at 18 months of age. During lung development various cellular and molecular events occur such as cell proliferation, cell death, differentiation and epithelial-mesenchymal transition (EMT). To understand the specific lethal events in Runx3 null mice, we examined cellular and molecular networks involved in EMT, and EMT inducers were quantified by RT-qPCR during lung development. Excessive EMT was observed in lungs at PN1 day in Runx3 null mice and PN18 months in Runx3 heterozygous mice. Pharmacologic inhibition of EMT was used to curb tumor progression. In this study, U0126 was injected to pregnant mouse for inhibition of pERK signaling. After U0126 treatment, life spans of newborn mice were increased and lung hyperplasia was partially rescued by down-regulated cell proliferation and EMT. Our data suggest that Runx3 is involved in crucial regulation of alveolar differentiation and tumor suppression in developing mouse lung.

Phosphorylation of the gastric tumor suppressor RUNX3 following H. pylori infection results in its localization to the cytoplasm

As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans‐differentiation of gastric cells to intestinal‐type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down‐regulated in most cases of IM without H. pylori‐infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori‐induced c‐Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3−/− mice expressed the intestinal markers Muc2 and Li‐Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori‐induced pathogenesis and the development of IM. J. Cell. Physiol. 227: 1071–1080, 2012.

RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction point

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 bound to its target loci, where it opened chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closed these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal was constitutively activated, RUNX3 inhibited cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

Abstract B35: Runx3 contributes for the defense against endogenous level of oncogenic K-Ras-induced tumorigenesis

Recent studies demonstrated that p53 affects only adenocarcinomas in K-RasG12V knockin mice leaving untouched adenomas which are likely to progress into adenocarcinomas (1, 2). These observations raised a fundamental question regarding whether cells can defend against endogenous level of oncogenic signal (3). Runx3, which plays pivotal roles in lineage determination and functions as a tumor suppressor, is frequently inactivated by promoter hypermethylation in various types of human tumors (4). Previously, we showed that deletion of Runx3 markedly enhanced K-Ras-G12V-induced lung tumorigenesis and led to early death (5). In this study, we found that immortalized Runx3-/- mouse embryonic fibroblast cells (MEFs) were tumorigenic. In addition, Runx3 was silenced by promoter hypermethylation in K-Ras-G12V-knockin MEFs. Expression of Runx3 in the K-Ras-G12V MEFs abolished their tumorigenesity. In mice, knockin of K-Ras-G12V in limited number of cells failed to induce tumor, but combination of K-Ras-G12V-knockin and Runx3-knockout by the same targeting method rapidly induced severe lung adenocarcinomas, lymphomas and skin carcinomas (median survival, 48 days). RUNX3 was silenced in all cases of K-Ras-activated human non-small cell lung cancers (n=20). Our results demonstrate that mammalian cells can defend against endogenous oncogenic K-Ras and Runx3 plays a key role in the defense mechanism. References 1. Feldser et al. (2010). Nature 468, 572-575. 2. Junttila et al. (2010). Nature 468, 567-571. 3. Berns A. (2010). Nature 468:519-20 4. Ito Y, Bae S-C, Chuang LS. (2015). Nat Rev Cancer. 15:81-95. 5. Lee et al., (2013). Cancer Cell. 24(5):603-16. Citation Format: Xin-Zi Chi, You-Soub Lee, Suk-Chul Bae. Runx3 contributes for the defense against endogenous level of oncogenic K-Ras-induced tumorigenesis [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B35.

Abstract A43: RUNX3 and pRB form a complex and regulate restriction-point commitment

Throughout the cell cycle, a cell monitors cumulative exposure to specific signals over time and makes a critical decision to pass through the restriction (R) point. Although the R-point decision is fundamental to normal differentiation and G1-S transition, the mechanism for the decision has been poorly understood. RUNX3 functions as a tumor suppressor and is frequently inactivated by DNA hyper-methylation in the stomach, bladder, colon, and lung. Recently, we have shown that adenovirus-Cre infected LSL-K-RasG12D mice induced lung adenocarcinoma (ADC) in prolonged latency (median survival, 220 days) and Runx3f/f/LSL-K-RasG12D mice rapidly induced ADC (median survival, 79 days), indicating a strong tumor suppressor activity of Runx3 against oncogenic K-Ras (Lee et., 2013. Cancer Cell. 2013 Vol. 24, pp 603-616). In the present study, we found that targeted disruption of Runx3 results in a delay in pRB phosphorylation and cell cycle progression through the R-point in mouse embryonic fibroblast cells. During the R-point interval, RUNX3 transiently formed a complex with pRB and BRD2 and induced Cdkn1a (p21Waf1/Cip1/Sdi1; p21). The complex was dissociated when by Cyclin D1 which was induced 4 hours after serum stimulation and p21 expression was turned off. However, oncogenic K-Ras maintained the complex. These results identify the series of molecular events associated with R-point commitment and provide an insight into the relationship between R-point commitment and oncogene surveillance. Note: This abstract was not presented at the conference. Citation Format: Xin-Zi Chi, You-Soub Lee, Jung-Won Lee, You-Mie Lee, Yoshiaki Ito, Suk-Chul Bae. RUNX3 and pRB form a complex and regulate restriction-point commitment. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A43. doi: 10.1158/1557-3125.RASONC14-A43

Abstract 2960: Identification of Runx3 as a gatekeeper of lung adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA It is thought that SP-C/CC10 double positive cells, which normally contribute to the renewal of lung epithelial cells, are the origin adenocarcinoma (ADC) of the lung. The present study shows that postnatal disruption of Runx3 in the mouse lung results in dedifferentiation of bronchiolar epithelial cells into SP-C/CC10 double positive cells. The double positive cells differentiate into SP-C single positive cells and adenoma (median survival, longer than 1 year). When Adenovirus-Cre was infected, LSL-K-RasG12D mice induced ADC in prolonged latency (median survival, 220 days) and Runx3f/f/LSL-K-RasG12D mice rapidly induced ADC (median survival, 79 days), indicating a strong tumor suppressor activity of Runx3. When Cre was activated in a limited number of cells through a leaky activation of Cretm/ERT in the absence of tamoxifen, K-RasLSL-G12D/+/Cretm/ERT mice did not induce lung ADC. In contrast, simultaneous inactivation of Runx3 and activation of K-Ras (Runx3f/f/K-RasLSL-G12D/+/Cretm/ERT) induced lung ADC with striking morphological similarities to human lung ADC in all mice (median survival, 48 days after birth). We also found that Runx3 forms a complex with BRD2 (a relative of TAF250) in a K-Ras activity-dependent manner and the complex transiently induces p14ARF and p21. When K-Ras was constitutively activated, the Runx3-BRD2 complex was maintained for long time and the expressions of p14ARF and p21 was continued. Disruption of the Runx3-BRD2 complex abolished expression of these two genes. These results suggest that cells recognize oncogenic K-Ras through a monitoring system composed of Runx3-BRD2 complex and Runx3-inactivation leads to abrogation of the monitoring system as well as dedifferentiation of lung epithelial cells. Citation Format: You-Soub Lee, Ju-Won Jang, Xin-Zi Chi, Yoshiaki Ito, Suk-Chul Bae. Identification of Runx3 as a gatekeeper of lung adenocarcinoma. [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 2960. doi:10.1158/1538-7445.AM2014-2960