The biology of tumors can only be understood by studying variant cell types within the tumor microenvironment (TME). The heterogeneity of tumors is based not only on the genomic profile but also on their microenvironment composition [14]. The microenvironment actively regulates tumor initiation, its progression, metastasis, and therapy response [15]. Hepatocarcinogenesis is a multifactorial process. Most HCC cases in western countries are associated with nonalcoholic steatohepatitis (NASH), alcohol abuse, hepatitis C virus (HCV), while in China, most with chronic infection with hepatitis B virus (HBV) inducing an inflammatory process followed by regeneration. Persistent concurrent regeneration and hepatic injury could produce an environment that eventually leads to the formation of hypoxia and inflammation, which are crucial features of HCC[16]. The inflammatory microenvironment facilitates the transformation of normal liver cells such as hepatocytes, immune, and stellate cells by providing a suitable environment for the development and progression of a tumor. Sustained inflammation followed by a continuous activation of immune cells can damage the DNA and bring about a neoplastic transformation. IL-22, one of the main T helper cell(Th17)-derived cytokines, has been linked as a vital element that allows tumorigenesis. Overexpression of IL-22 has been observed in several human tumors, such as liver, breast and prostate cancers. It has the capacity of prompting cell differentiation and proliferation, and, in mice models, some reports suggest that IL‐22 favors tumor metastasis in lung, colon cancers and non‐melanoma skin cancer[17]. In humans gastric, colorectal and pancreatic cancer, elevated expression of IL‐22 and IL-22 receptor were reported to correlate with disease progression and poor overall survival[18, 19]. It has also been observed that patients with liver fibrosis and advanced cirrhosis present high serum levels of IL-22. Consistent with our previous study, we further verified the inhibitory effect of metformin on HCC by using DEN-induced mouse model. This model mimics aspects of liver injury, hepatitis and fibrosis, which all are the basis of human HCC[20]. Despite current findings, transgenic murine models with IL-22 overexpression and knockout are warranted to investigate to achieve an even convincing agreement.
Once a tumor is established, various cytokines can be recruited from a distant place of the same organ or peripheral tissues into the TME. The persistent inflammatory milieu not only promotes tumor development but also accelerates tumor progression, stimulates invasion, angiogenesis, and metastasis through the release of several mediators. Our data indicate that IL-22 is one of these crucial mediators for tumor progression and metastasis. The results are in accordance with Jiang et.al [13]which shown IL-22 had a prominent effect on tumor cell survival, proliferation, invasion, metastasis, as well as malignancy transformation from chronic hepatitis. It’s easy to understand that any changes in the microenvironment could support the development of HCC, and the complexity of TME and therapeutic failures may be explained, in some degree, by alterations of components of the TME. Metformin was introduced into HCC treatment in our study and had shown to restrain IL-22 related tumorigenesis and progression both in vitro and in vivo.
However, HCC microenvironment is composed of numerous tumoral and non-tumoral cell types, and cytokines and other components that are in continuous interaction and communication with each other. Their interactions make an important contribution to tumor progression by modulating tumor cell properties. This scheme is far more complex and requires orchestrated regulation. In addition, functions of numerous cytokines secreted by diverse immune cells upon certain stimuli may overlap. The intricacy of the system means, on the other side, that effect of metformin on one single cytokine within tumor tissue extremely depends on the context of the individual microenvironment and is hard to predict. The major challenge of future studies remains to predict the role for an individual response in any given microenvironment.
MHCC97-H and SMMC-7721 are two types of human HCC cell lines with varying biological behaviors. MHCC97-H cell is characterized by larger cell volume and a highly invasive phenotype[21].SMMC-7721 cell, originated from a Chinese HCC patient, is widely used in the HCC research concerning anticancer effects and mechanisms of various anti-cancer drugs[22]. In the present study, IL-22 promoted cell proliferation, survival, metastasis and invasion compared with control cell lines, whereas the disadvantage was significantly undermined by metformin treatment. This result was confirmed in vivo using subcutaneous xenograft model. The underlying mechanism probably involved Hippo pathway. However, it should be noted that heterogeneity between HCC cells need to be taken into account. Further studies with more different HCC cells or tissues can be more informative.
The Hippo signaling pathway, an highly conserved signaling pathway regulated by Mst1/2, controls organ size, tissue regeneration, as well as tumor progression through the regulation of cell apoptosis and proliferation[23]. YAP, a protein that acts as a transcriptional regulator by activating the transcription of genes involved in cell apoptosis and proliferation, is the main downstream effectors of the Hippo signaling pathway[24].CYR61 and CTGF, direct targets of YAP, belong to extracellular matrix -associated signaling protein of the CCN family which are capable of regulating a broad range of cellular activities, such as cell adhesion, proliferation, senescence and apoptosis[25]. Given that they play a positive role in cell proliferation, it’s not surprising that these two proteins have been found to be elevated in some human cancers, including pancreatic malignancy[26], gliomas[27], prostate cancer[28] and breast cancer [29]. Abnormal activation of TAZ and YAP caused by aberrant Hippo signaling is continually observed in many human cancers [30]. Yap ectopic expressed specifically in liver can result in a significant increase in size. Remarkably, the liver reverts back to its properly size when Yap overexpression is turned off [31]. Similarly, enlargement of liver is also reported in Mst1/2 knockout mice[32]. These observations suggest that YAP/TAZ are identified as oncogenes involved in the Hippo signaling pathway. Importantly, activation of YAP as a consequence of abrogating Hippo signaling by knocking out Mst1/2 was sufficient to drive liver carcinogenesis in mice[33]. Interestingly, we found that IL-22 transfected tumor cell resulted in inactivation of Hippo signaling as shown by lower expression of Mst1/2 and afterwards phosphorylation of YAP which leads to the suppression of YAP activity. Meanwhile, metformin dramatically impaired this tumorigenesis effect. The mechanism of which metformin regulates the Hippo pathway could be attributed to activation of AMP-activated protein kinase (AMPK)[34]and this makes our results more understandable. However, it should be noted that many factors, such as proteins that determine cell adherens, tight junctions and polarity, can contribute to regulation of Hippo signaling pathway. Thus, it still remains to examine further possible mechanisms involved in the presence of metformin, on the other hand, a similar experimental design with Mst1/2 knockout mice is warranted to investigate the therapeutic potential of metformin in the prevention of HCC.
Given that TME is deeply involved in many human cancers, there is a growing enthusiasm in developing drugs that may affect it as a promising cancer treatment. The data from mouse models and cell experiments studied in this work raise the possibility that aiming at TME metformin could have desirable antitumorigenic effects, however, its clinical utility is yet to be ascertained.