HIC1 attenuates invasion and metastasis by inhibiting the IL-6/STAT3 signalling pathway in human pancreatic cancer

Highlights

• Expression of HIC1 is silenced in PDAC, which predicts worse patient survival and prognosis.

• HIC1 inhibits pancreatic cancer invasion and metastasis.

• HIC1 impairs STAT3 DNA binding activity and represses STAT3 target genes expression.

• HIC1 and STAT3 interaction is very important to elucidate the mechanism of pancreatic cancer invasion and metastasis.

Abstract

Hypermethylated in cancer 1 (HIC1) is a tumour suppressor gene that is frequently deleted or epigenetically silenced in many human cancers. However, the molecular function of HIC1 in pancreatic cancer has not been fully elucidated, especially in cancer invasion and metastasis. We aimed to clarify the clinical relevance of HIC1 and human pancreatic cancer and the mechanism of its effect on invasion and metastasis .HIC1 was downregulated in pancreatic cancer patient cancer tissue and pancreatic cancer cell lines. A tissue microarray analysis demonstrated that negative HIC1 expression predicted advanced pathological stages and worse patient survival. In addition, HIC1 inhibited the invasion and metastasis of pancreatic cancer cells both in vitro and in vivo. Finally, HIC1 repressed the expression of STAT3 target genes, including c-Myc, VEGF, CyclinD1, MMP2 and MMP9, by binding and interacting with STAT3 to impede its DNA-binding ability but without affecting the protein levels of STAT3 and p-STAT3. Therefore, HIC1 appears to function as a STAT3 inhibitor and may be a promising target for cancer research and for the development of an optimal treatment approach for pancreatic cancer.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death for patients in the United States, with a median survival of less than six months and a dismal five-year survival rate of < 5% upon diagnosis [1], [2]. PDAC is notorious for its propensity for early lymphatic invasion, easy liver metastasis, rapid recurrence, and poor prognosis [3], [4], [5]. Surgery is considered the only method for radical treatment, but most patients have missed the opportunity for surgery by the time of diagnosis. This aggressive biology and resistance to conventional and targeted therapeutic agents leads to a typical clinical presentation of incurable disease [1], [6], [7]. However, the lethal nature of pancreatic cancer and the mechanism underlying its rapid dissemination to the lymphatic system and distant organs are poorly understood and not fully elucidated [1], [3], [8]. The process of PDAC metastasis involves genetic changes (k-ras, p16, p53, smad4, etc.), the tumour microenvironment (TME), various molecular components (SP1, AP1, VEGF, c-Myc, MMPs, Snail, Twist etc.), and the activation of relevant signalling pathways [7], [9], [10], [11], [12], [13].

Current studies identify signal transducers and activators of transcription-3 (STAT3) as a central regulator of tumour metastasis [6], [14]. The pivotal role of STAT3 was highlighted in cancer metastasis, leading to therapeutic strategies that target the STAT3 signalling pathway for the inhibition of metastasis [14], [15], [16]. STAT3 is well known to be activated by numerous cell cytokine receptor-associated tyrosine kinases (interleukin-6, IL-6/IL-6R), growth factor receptors with intrinsic tyrosine kinase activity (EGF/EGFR, FGF/FGFR, PDGF/PDGFR, VEGF/VEGFR), and oncogenic proteins (SRC and ABL), which suggests that STAT3 signalling is one of the most important pathways commonly involved in regulation of cancer invasion and metastasis [6], [9], [11], [17], [18]. However, the IL-6/JAK/STAT3 signalling pathway was also illustrated to govern PDAC metastasis [10], [11]. The IL-6/JAK/STAT3 pathway is frequently constitutively activated in PDAC tissue specimens obtained from pancreatic cancer patients undergoing surgery and in pancreatic cancer cell lines [9], [11], [17], [18]. A number of genes downstream of the activated STAT3 pathway are closely related to PDAC invasion and metastasis, including tumour angiogenesis agonists (VEGF, COX-2), cell-cycle regulators (c-Myc and CyclinD1), apoptosis inhibitors (Mcl-1 and Bcl-xL) and matrix metalloproteinases [3], [14], [15], [17], [19].

Hypermethylated in cancer 1 (HIC1) is a tumour suppressor gene located at chromosome 17p13.3 that is rich in CpG islands and frequently deleted or epigenetically silenced in a variety of human cancers, including breast cancer, liver cancer, prostate cancer, lung cancer, colorectal cancer, leukaemia and pancreatic cancer [20], [21], [22], [23], [24], [25], [26]. HIC1 exerts broad biological functions during normal development and is implicated in many canonical processes of carcinogenesis, such as the control of cell growth, cell survival, cell migration, and motility [17], [24]. Recently, downstream target genes of HIC1 responsible for developmental, proliferation, migration, invasion, angiogenesis and cell-cycle control have been identified, including the SIRT1, ATOH1, TCF4, CXCR7, CyclinD1, P57^KIP2, ephrin-A1, Eph A2, SOX9, and FGF-BP1 [17], [20], [21], [25], [26]. However, few HIC1 target genes have been characterized. Nevertheless, the specific and particular molecular mechanism played by HIC1, especially in cancer invasion and metastasis, have not been fully elucidated. Therefore, our attention was drawn to a recent report describing the effect of HIC1 on the activity of STAT3 [27].

In this study, we detected both the mRNA and protein levels of HIC1 in pancreatic cancer patient samples and PDAC cell lines. We also analysed the correlation between the HIC1 level and patient survival and prognosis. Furthermore, we examined the effect of the restoration of the HIC1 protein level on pancreatic cancer invasion and metastasis in vivo and in vitro. Finally, to explore the mechanism of this effect, we observed the interaction between HIC1 and STAT3 and then evaluated the impact of this interaction on the activation of the IL-6/JAK/STAT3 signalling pathway as well as the downstream target gene expression.