Intrahepatic cholangiocarcinoma arises from epithelial cells lining the bile duct and occurs proximally at the second degree bile ducts within the liver. The condition is commonly associated with cirrhosis, viral hepatitis B and C [1]. ICC displays a feature of rapid progression and a poor outcome, and its global disease incidence has been rapidly increasing [2]. Radical resection and curative liver transplantation are preferred surgical treatments for ICC, however, the patients with vascular and lymph nodes metastases are not eligible for surgical therapy. Although the chemotherapy regimen of gemcitabine and cisplatin and locoregional therapies are additional options for inoperable ICC patients, the 5-year survival rates are very low. An enhanced understanding of the biology pathological progress and the interaction with tumor microenvironment of ICC is needed to improve patient survival.

PTHLH, also referred to parathyroid hormone-related protein (PTHrP), has emerged as an important cytokine with diverse cell functions, including growth, survival, migration, and differentiation [3]. Tumor-derived PTHLH participates in the bone metastatic processes of breast cancer via an intracrine fashion [4]. In addition, PTHLH supports colorectal cancer cell to form distant lung metastatic processes via inducing caspase-independent death in human lung vasculature endothelial cells [5]. A previous report demonstrated that PTHLH produced by proliferating bile duct epithelial cells and may interact with growth factors and hormones to form complex loops that promotes proliferation [6]. Growing evidences indicate that PTHLH-producing cholangiocarcinoma (CHO) patients suffer from humoral hypercalcemia of malignancy [7–9], but litter is known regarding PTHLH’s effect on ICC cells growth. MAP kinase pathways are involved in the process through the PTHLH-induced activation of PTH1R to activate downstream effectors [10–12]. ATF2, as a downstream effector of MAPK in response to cytokines, is phosphorylated on Thr69 and/or Thr71 by either JNK or p38 [13, 14], and is also activated by the ERK1/2 pathway in two step [13]. Several observations support that ATF2 regulates cell cycle progression via controlling the transcriptional output of several key genes, including CyclinD1, CyclinA and RB1 [15–18]. However, no studies have documented a role for the PTHLH-MAPK-ATF2-CyclinD1 signaling axis in the regulation of ICC cells growth. This study aims to elucidate the role and clinical significance of the PTHLH-MAPK-ATF2-CyclinD1 axis in ICC cell cycle progression.

ICC is one of the most lethal epithelial cancers united by poor diagnoses and adverse outcomes. The condition frequently arises in the presence of chronic injury and inflammation. Previous literatures documenting that ICC is commonly associated with cirrhosis, viral hepatitis B and C, and metabolic abnormalities [19–22]. The molecular pathogenesis of ICC proliferation and metastasis, as the main cause of ICC-related mortality, but the mechanisms remain obscure. Our findings demonstrate that PTHLH knockdown in ICC cells suppressed tumor growth, while re-expression of PTHLH has the opposite effect, highlighting the role of PTHLH as a critical oncoprotein in ICC progression.

PTHLH/PTH1R signaling is aberrantly induced or activated in different cancer types and is associated with poor prognosis [5, 23–25]. In the present study, we found that ICC cells produced PTHLH ligands that respond via the expression of cognate receptors PTH1R, resulting in the continuous activation of downstream signaling pathways. Extensive evidence suggest that PTHLH is viewed as a cellular cytokine, particularly in epithelial cancer cells, that exhibits an autocrine or paracrine role in both cell growth and differentiation [26, 27]. In our previous study, we found that overexpression of the PTHLH (LV-PTHLH), which transfects lentivirus-mediated PTHLH_GFP without deleting endogenous PTHLH expression in ICC cells, may enter into the non-proliferative cells (data not show). In contrast, LV-PTHLH_RE ICC cells promoted cell growth. Moreover, endogenous PTHLH sustains the activation of MAPK signaling pathways, and this effect was more pronounced after the addition of a PTHLH recombination peptide. In contrast, the effect of PTHLH on activation was attenuated at a higher concentration (100 nM) compared with 50 nM (Fig. 5b, dotted portion). These results clearly indicated that PTHLH may function as a tumor cell growth promoter within a certain concentration range. When the range is exceeding, PTHLH becomes saturated and suppresses cells proliferation. In our study, we also found re-expression PTHLH promotes RBE cells migration and specific overexpression PTHLH associates with intrahepatic metastasis in ICC patients. All results indicated a potential ability of facilitating tumor invasiveness. The data would suggest that PTHLH may potentially transform ICC cells into an aggressive form of the disease. And this also indicates that PTHLH influences ICC cells growth and differentiation with a low signal expression.

To investigate the effect of ICC secreted PTHLH on cancer cell growth, we established an in-vitro PTHLH secretion system. In our present work, we found that PTHLH regulated cell growth by altering the cell cycle. Cell cycle dysregulation is a major feature of tumorigenesis, which occurs by shortening the G₁ phase or activating CDKs may favor tumor development [28]. Herein, we demonstrate that PTHLH controlled cell cycle progression. Secreted PTHLH protein act on target cells by binding to its specific cell surface receptor: PTH1R. The potential molecular mechanisms could be explained by the finding that the PTHLH protein activates the JNK/ERK1/2-ATF2 axis via interacting with the MAPK scaffolding protein β-arrestin2, or triggering an early G protein-dependent pathway meditated by PKA and PKC [10] leading to cell cycle proliferation. Furthermore, we provided evidence suggesting that PTHLH upregulates ATF2 phosphorylation via activating the ERK1/2 and JNK signaling pathways, which transcriptionally upregulate CyclinD1 expression. When ERK1/2 and JNK are pharmacologically inhibited in ICC cells (i.e., via U0126 and SP600125), which blocked PTHLH-induced activation of ERK1/2 and JNK signal pathways, and transcriptional activity of ATF2 (Fig. 6). Cyclin D1 is frequently deregulated in cancer and is a biomarker of cancer phenotypes and disease progression [29]. Previous findings suggested that the deregulation of CyclinD1 expression and CDK4 activation directly lead to some cancer hallmarks by inducing proliferation [30–32]. We observed that reduced CyclinD1 expression and CDK4 inactivation directly inhibited proliferation, which is consistent with previous reports. Consistently, our present work demonstrated that PTHLH re-expression accelerated the G₂ to M phase transition, which is similar to the effects of numerous other oncogenes. Among the genes functioning during the G₂ and M phase transition, we observed that the p-cdc2 levels were rapidly reduced upon PTHLH re-expression. Several experimental findings indicate that cdc2 is one of the master regulators of mitosis that controls the centrosome cycle in complex with A- or B-type cyclins [33]. Previous reports have suggested that cdc2 activity upon mitosis entry depends on p-cdc2 levels [34]. However, the reduction of cdc2 activity primarily drives the exit from mitosis [33]. Our present study indicates that PTHLH promote mitosis in ICC cells via downregulating p-cdc2 expression. Interestingly, we observed a paradoxical phenomenon that PTHLH overexpression without knocking-down endogenous secretion arrests ICC cells in the G₁ phase and decreases CyclinD1 expression (data not show). We hypothesized that ostensibly paradoxical responses between PTHLH deletion and overexpression in cultured ICC cells appear to facilitate a compromise between maximal mitogenic stimulation and the avoidance of antiproliferative defenses.

Another interesting finding of this study is the negative regulated role of ATF2 in RBE cells (Fig. 6). ATF2 is an important transcription factor that can facilitate malignant proliferation. In our previous study, we found that ATF2 promoted growth of ICC cells and was correlated with a poor prognosis for ICC patients. In vitro evidence indicated that the upregulation of ATF2 phosphorylation and activity promotes cancer progression via facilitating cell proliferation-related gene expression. In our study, we demonstrated that PTHLH can promote oncogenic functions of ATF2 by activating ERK/JNK pathways. And a previous report revealed PTHLH can activate PKC pathway [35]. Eric Lau [36] previously reported that PKC_ε promotes oncogenic functions of ATF2 in the nucleus while blocking its apoptotic function at mitochondria. Thus, we believe that PTHLH promotes nuclear translocation and transcriptional function in the oncogenic functions of ATF2. Interestingly, another novel finding of our study is the negative role of ATF2 in PTHLH production in REB cells. And we also found autocrine activation of the PTHLH promoter by c-Jun (data not show). Collectively, these findings indicated that ATF2 limits PTHLH transcriptional output to maintain specific concentration by forming a homodimer or a heterodimer with JUN. It is possible that cancer cells may have negative feedback loops that are essential for survival.

In summary, we report that ICC-secreted PTHLH acts in an autocrine manner in intrahepatic cholangiocarcinoma progression by activating the canonical ERK/JNK signaling pathway. However, our findings focus on only PTHLH-mediated ICC cell proliferation, not provide new insight into the ICC metastasis. Despite the importance of PTHLH tumorigenic role, our knowledge of the PTH1R that mediate changes in the tumor progression and interaction with PTHLH in ICC is still limited. Based on our findings, further investigation for interfering with PTH1R, which mediate signaling in cancer cells, may serve as effective treatment approaches to ICC patients. And we will improve the mechanisms of PTHLH/PTH1R-mediated ICC progression and involve the interaction of the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) in ICC development.