RUNX3 suppresses metastasis and stemness by inhibiting Hedgehog signaling in colorectal cancer

Abstract

Disabled tumor suppressor genes and hyperactive oncogenes greatly contribute to cell fates during cancer development because of their genetic alterations such as somatic mutations. However, little is known about how tumor suppressor genes react to diverse oncogenes during tumor progression. Our previous study showed that RUNX3 inhibits invasiveness by preventing vascular endothelial growth factor secretion and suppressed endothelial cell growth and tube formation in colorectal cancer (CRC). Hedgehog signaling is crucial for the physiological maintenance and self-renewal of stem cells, and its deregulation is responsible for their tumor development. The mechanisms that inhibit this pathway during proliferation remain poorly understood. Here, we found that the tumor suppressor RUNX3 modulates tumorigenesis in response to cancer cells induced by inhibiting oncogene GLI1 ubiquitination. Moreover, we demonstrated that RUNX3 and GLI1 expression were inversely correlated in CRC cells and tissues. We observed a direct interaction between RUNX3 and GLI1, promoting ubiquitination of GLI1 at the intracellular level. Increased ubiquitination of GLI1 was induced by the E3 ligase β-TrCP. This novel RUNX3-dependent regulatory loop may limit the extent and duration of Hedgehog signaling during extension of the tumor initiation capacity. On the basis of our results, identification of agents that induce RUNX3 may be useful for develo** new and effective therapies for CRC.

Introduction

The development of tumor cells often results from multiple genetic alterations that cause a cellular shift. Numerous specific genetic alterations have been identified that activate proto-oncogenes or inactivate tumor suppressor genes. Indeed, the sine qua non of a tumor gene is that it is affected by a mutational event with a vital prevalence. In addition, a ‘third’ pathway to tumorigenesis has been identified whereby the expression of key genes is regulated through promoter hypermethylation and silencing. Particularly, tumor suppressor genes may be subject to this mechanism of inactivation, in addition to mutational events. Oncogenes and tumor suppressor genes have classically been assigned distinct, independent roles in tumor progression. However, the relationships between these tumor-promoting processes remain poorly understood.

The human RUNX gene is homologous to the Drosophila genes Runt and Lozenge [1, 2] and encodes a subunit of the Runt-domain transcription factor PEBP2/CBF [3]. The Runt-related transcription factor (RUNX) family includes RUNX1, RUNX2, and RUNX3, which play a role in cell proliferation and differentiation in humans [4, 5]. RUNX3 has been shown to act as a tumor suppressor in gastric cancer [6], and previous studies have indicated that it is downregulated in various tumors [7]. Moreover, inactivation of RUNX3 is caused by promoter hypermethylation, loss of heterozygosity, or mislocalization [8, 9]. Various studies have demonstrated that RUNX3 can function as a tumor suppressor by regulating metastasis in cancer [44, 45]. The stability of GLI1 via SHh signaling is associated with a malignant phenotype in various cancers. In addition, it has been reported that increased GLI1 enhanced tumor induction in transgenic mice [46]. Unlike SHh and IHh signaling regulated GLI1 through interactions with RUNX2 and RUNX3 in a chondrogenesis mouse model [47].

Our study investigated the possibility of using RUNX3 and GLI1 as biomarkers in the patients with CRC. We demonstrated a difference of survival by expression of RUNX3 in early stages such as stages I and II. Typically, patients in these stages are not administered adjuvant chemotherapy after corrective resection of the primary tumor and show a high recurrence rate. Clinical biomarkers that detected poor prognosis patients are urgently needed.

In summary, we showed that Hh-GLI signaling, a major regulator of tumorigenesis, is suppressed by RUNX3. This phenomenon involves the ubiquitin-regulated processing of GLI1, which is mediated by functional cooperation between RUNX3 and the E3 ubiquitin ligase β-TrCP (Fig. 6g). RUNX3 also regulates the transactivation of Notch1 and, consequently, the cell proliferation fate by Notch signaling [48]. Functional crosstalk between the Notch and Hh pathways is known to occur during development and tumorigenesis, but the underlying mechanisms are unclear. Ultimately, considering the roles of Hh signaling in tumorigenesis, appropriate molecular targets should be considered for evaluation in clinical anticancer drug trials focusing on Hh signaling suppression. Our founding improves the understanding of the mechanism by which connecting to RUNX3 as a tumor suppressor and GLI1 as an oncogene occurs as it relates to the metastatic and drug resistance of CRC.