Over the last few decades, various tumor markers have been used in human and veterinary medicine for the diagnosis, prognosis, and monitoring of cancer cases. Identifying new molecules and signaling pathways involved in lung carcinogenesis is still required for treatment outcomes and prognosis.
In human medicine, pulmonary cancer is classified into small-cell lung cancer (SCLC), originating from bronchial neuroendocrine cells, and non-small cell lung cancer (NSCLC), originating from bronchoalveolar epithelial cells [31]. The most common type of NSCLC is represented by adenocarcinoma, accounting for 40% of cases [32, 33]. In sheep, OPA lesions are those of a well-differentiated bronchoalveolar adenocarcinoma with a papillary or acinar predominant growth pattern [34, 35]. In our cases, similar results were observed and the predominant histological type was represented by acinar.
The main signaling pathways involved in human lung cancer include the following: growth-promoting pathways (EGFR/Ras/PI-3-Kinase), growth inhibitory pathways (p53/Rb/P14ARF,STK11), apoptotic pathways (Bcl-2/Bax/Fas/FasL), DNA repair and immortalization genes [36–40]. In OPA, the Ras-MEK-ERK, PI3K-AKT-mTOR, EGFR, RON-HYAL2, and heat shock proteins are described as commonly activated pathways [41, 42]. Recently, increased gene expression of anterior gradient 2 (AGR2), amphiregulin (AREG), yes-associated protein 1 (YAP1) has also been reported, suggesting a role for Hippo pathway. However, significant differences have been observed between natural and experimentally infected cases [43]. However, in comparison to human lung cancer, an important and unique aspect of JSRV biology is the env protein that directly activates cellular signaling mechanisms leading to neoplastic transformation and proliferation [4, 6, 44]. Thus, JSRV is unique due to the env protein being directly oncogenic, as opposed to other viruses that cause neoplastic transformation by insertional mutagenesis or oncogene capture [4, 45]. Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway plays an important role in several physiological processes, such as immunity, cell growth, and differentiation [46]. The important role of the IL-6-STAT3 pathway has been intensely studied in human cancers, particularly in lung tumors [47, 48]. It has been demonstrated that STAT3 has multiple effects in pulmonary cancer including preventing apoptosis, promoting cell proliferation and angiogenesis, and helping tumor cells to evade anti-tumor immunity JAK/STAT3 signaling is commonly activated by IL-6 and dysregulation of the interleukin IL-6-mediated JAK/STAT3 signaling pathway is closely related to the development of various human tumors and is often associated with poor patient outcomes [49]. According to the authors’ knowledge, the IL-6 and STAT3 expression has not previously been evaluated in spontaneous OPA cases.
This study evaluated the immunoexpression of IL-6 and STAT3 in two groups of sheep pulmonary tissues: 1) healthy tissue (control group); and 2) neoplastic lesions (OPA group). By immunohistochemistry, IL-6 expression was observed in the neoplastic epithelial cells in 19 of 20 (95%) OPA cases, while STAT3 immunolabeling was demonstrated in all pulmonary adenocarcinomas (100%). In the healthy tissue control group, scattered epithelial cells showed weak expression of both IL-6 and STAT3. A statistical correlation was not performed between the control and OPA groups because the counting error for type II pneumocytes was considered high, in the absence of the double immunohistochemistry technique.
Furthermore, Western Blot analyses revealed upregulation of IL-6 (p = 0.0078) and STAT3 (p < 0.0001) in neoplastic tissue by comparison with normal lung tissue. This technique is based on molecular weight, which aims to identify specific proteins from a complex mixture extracted from cells [50]. Western blot analysis revealed bands at the appropriate molecular weight for both IL-6 and STAT3 (25kDa and 90kDa respectively), indicating that the two antibodies used in this study are correctly reacting with this species.
IL-6 participates in numerous signaling pathways one of which is Janus kinase (JAK) [51]. JAK is activated by ligation of IL-6 with IL-6R causing phosphorylation of STAT3. This phosphorylation starts homodimerization and allows STAT3 to enter the nucleus, where it affects the expression of STAT3 target genes, which encode proteins that play a role in cell proliferation (cyclin D1), and/or their survival (BCL2-like protein 1) [52]. STAT3 also induces IL-6 expression, thus causing a cyclic autocrine feedback mechanism [53].
Sources of IL-6 are variable, but in a neoplastic process, IL-6 is produced by several cell types including stromal cells, inflammatory cells, and neoplastic cells. In inflammatory processes, the main sources are macrophages (acute inflammation) and T cells (chronic inflammation) [24, 31, 46, 50, 54–56]. High IL-6 serum levels were reported in human patients with pulmonary tumors including NSCLC [16, 57]. In the present study, we were able to show immunohistochemically that OPA neoplastic epithelial cells are also capable of producing IL-6. In addition to the neoplastic epithelial cells, IL-6 was also produced by alveolar macrophages within perineoplastic areas, intratumoral inflammatory infiltrates, as well as neoplastic stromal tissue. Alveolar histiocytosis is a commonly observed feature in OPA, and the influx of macrophages is most likely the first event following JSRV infection [37]. Even though distinguishing macrophage subtypes was not an explicit objective of this study, alveolar and intratumoral macrophages frequently immunoexpressed IL-6, suggesting that these macrophages may also have an important role in IL-6-STAT3 expression in OPA cases. Recently, numerous genes related to macrophage immunoregulatory function were found to be activated in experimental and natural cases of OPA, suggesting an important role in the pathogenesis of the disease [43].
Previous studies of experimental OPA carcinogenesis showed that by applying JAK inhibitors, the neoplastic transformation of fibroblast cell cultures by JSRV env protein was not affected.[56] Western Blot analyses presented in this study showed significantly higher levels of IL-6 and STAT3 in the OPA group compared to the control group, and immunohistochemically the neoplastic cells expressed both markers. One possible explanation might be a difference in the microenvironments of cell culture versus cancer, and as mentioned earlier, IL-6 is produced in large amounts by inflammatory cells within the tumor microenvironment.
An increased STAT3 activity represents a negative prognostic marker in human neoplasms including lung cancer, particularly NSCLC.[58, 59] Therefore, the possibility of targeting the STAT3 pathway as a possible therapy for treating such tumors is the subject of an ongoing investigation.
The main limitation of this study is the lack of assessment of the phosphorylated variant of STAT3. However, the STAT3 antibody labels the total STAT3, and moreover, nuclear staining was observed in all cases from the OPA group, suggesting activation and translocation of STAT3 to the nucleus. Another limitation is the low number of animals in the study group. Only 20 animals with OPA were available. However, OPA is only present at a low prevalence rate in countries where it is present. The previously reported prevalence of affected animals within the Romanian sheep population is 1.26% [5]. Similar study regarding the prevalence of OPA in sheep population are reported in other European countries. For instance, the prevalence of OPA in the United Kingdom in 2015 was reported as 0.9% and was reported as 0.5% from 369 animals tested in the Republic of Ireland [60, 61].