Analyzing the influence of IL18 in regulation of YAP1 in breast oncogenesis using cBioportal

Abstract Background Yes‐associated protein 1 (YAP1) is responsible for tumor growth, progression and metastasis. The mechanisms controlling the generation and relative ratio of the functional YAP1 and other co‐factors are not well‐understood. Various literature reported that co‐factors like cytokines significantly influence signaling pathways to introduce epithelial immunity and regeneration, which later helps increase cancer‐related phenotypes. Among various cytokines, IL‐18 has emerged as a major player in inflammation and progression of different types of cancers. Till now, much information has not been known about the role of YAP1 in tumor aggressiveness and immune evasion in breast cancer with respect to IL‐18. Aim We aimed to explore the effect of YAP1 in tumor aggressiveness and immune evasion in breast invasive carcinoma and metastatic breast cancer in the context of Interleukin‐18 (IL‐18) in silico. Methods and Results We used publicly available data generated by The Cancer Genome Atlas (TCGA) Research Network through cBioportal web platform. Kaplan–Meier method was used to determine the overall survival and comparison between curves were made using Log‐Rank test. The p values were determined by Fisher's exact test with the null hypothesis. Correlation plots were analyzed by comparison with gene copy numbers from the GISTIC2.0, available through cBioportal. Our analyses suggest that IL‐18 influences YAP1 expression in breast oncogenesis via Interferon‐gamma (IFN‐γ) production. Patients having a higher expression of IL‐18 possess a better prognosis and higher YAP1 expression with lower IL18 drives to poor clinical results in breast cancer. Conclusion This can provide new approaches to better understand the relation between YAP1 and IL‐18 in breast cancer progression by performing in vitro and in vivo studies. Also, IL‐18 can be considered as a potential target for tumor treatment in YAP1 overexpressed breast carcinoma.


| INTRODUCTION
Progressive immune dysregulation, occurring at different levels, contributes to uncontrolled tumor growth and eventually, cancer progression. A primary purpose of Cancer Immunology Research Program is to single out the proper mechanisms of anti-tumor immunity that could instruct the development of unique and effective immunetherapies. It is well evident that the functional status of immune system has immense direct comportment on breast cancer. However, the proper mechanisms behind breast cancer pathophysiology are still not well defined. 1 Since most of the genes in murine models, including diseases causing, are very close to human genes, it helps intensely to understand the mammalian, especially the human innate immune responses. 2 The ability to defend against infection and destroy cancerous cells depends on lots of intracellular factors, among which the hippo signaling pathway is among the most important ones. 1 It plays an important role in controlling cell size and number through proliferation and apoptosis. 2 At the center of the Hippo cascade in human, is the transcription factor yes associated protein-1 (YAP1). When activated, YAP1 translocates into the nucleus and binds transcription enhancer factors to promote the transcription of genes regulating proliferation. 2, 3 Guo et al 4 described that YAP1 suppresses cell apoptosis and encourages cell proliferation in breast cancer through the phosphatase and tensin homolog deleted 10-AKT signaling pathway.
There are several reports which indicates that, YAP1 plays an important role in other types of cancers too. [5][6][7] Nonetheless, the mechanisms controlling the generation and relative ratio of the functional YAP1 and other co-factors are not wellunderstood. Among those co-factors, cytokines are the most important groups. Various literature reported that cytokines significantly influence signaling pathways to introduce epithelial immunity and regeneration, which later helps increase cancer-related phenotypes. 8 Cytokines belong to a group of low-molecular-weight polypeptide proteins and are well known for their invaluable role in inflammation and immune response regulation. 9,10 Production of abnormal cytokines and/or their receptors can result in inflammatory diseases and cancers. Some cytokines, like interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a), are secreted during hypoxia, which is a hallmark of tumor. In inflammation and breast cancer, the role of cytokines is being investigated thoroughly. 2,3 Also, cytokines play a crucial role in the entangled link between tumor cells and tumor-infiltrating innate and adaptive immune cells inside the tumor microenvironment. 8,9,11 These tiny molecules get involved in the specific cellular functional mechanism, thus regulating signaling pathways to induce cancer. 8,10 Cytokines help to recruit immune cells to local inflammatory sites, [12][13][14] resulting in the enhancement of tumor recognition by immune cells. 15,16 Cytokine molecules like IL-1α, IL-1β, IL6, IL-10 inhibit and eliminate cancer [17][18][19] by infiltration of immune cells in local tumor masses, 20,21 enhancing tumor immunoediting properties, [22][23][24][25] patronage tumor invasion and metastasis. [26][27][28][29][30] Several reports show that cytokines take part in integrating breast cancer commencement and progression. [31][32][33][34] Till now, functions of cytokines and cytokine receptors have been vastly studied in murine models and to some extent in humans; a need remains for well-thought-out research about the contribution of cytokines in inflammation and human cancers.
Inflammation is a consequence of the defensive reaction of the body against diverse pernicious stimuli. However, the production of abnormal cytokines and their receptors can result in inflammatory diseases and cancers. [35][36][37] While the profound inflammatory reaction is likely to resolve once the affront entity is decreased, this otherwise sudden and short-term response becomes long-lasting when the body fails to neutralize the inflammatory reactions. The inflammatory microenvironment is correlated with the secretion of various types of pro-inflammatory and oncogenic molecules like interleukin-1 beta (IL-1β), interleukin-2 (IL-2), interleukin-6, interleukin-18 (IL-18), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), several growth factors and chemokines. 38,39 Among various cytokines, IL-18 has emerged as a major player in inflammation and progression of different types of cancers. [40][41][42][43] This cytokine was first discovered as an inducing agent of IFN-γ in the mice sera, injected with endotoxins. 2 Different types of activated immune cells are responsible for the production of this cytokine. Among them T cells, B cells, dendritic cells, natural killer cells, macrophages and neutrophils are most important. 44 IL-18 induced activated T helper 1 (Th1) cells produce IFN-γ which enhances lymphocyte proliferation, thus play a crucial role in innate and adaptive immunity regulation. 45 Considering the host environment, IL-18 actively takes part in the inflammatory response and immune escape of neoplastic cells. 9,46 A previous study showed that IL-18 plays both pro and antiinflammatory roles in cancer progression. 9 Yang et al 14  hence, it can be crucial in metastasis and pathogenesis in breast cancer. But on the other hand, another study showed that IL-18 and B7-1 molecules together increase cytolytic activity in vivo by infiltrating natural killer cells into tumors by recruiting IFN-γ, thereby suppressing lung metastases and prolong survival. 47 Chang et al 48 studied recently that IL-18 DNA, while injected intratumorally, increases the production of IFN-γ and subsequently suppresses the liver tumor. Another study revealed that YAP1 with telomere dysfunction is involved in increased production of IL-18 through the engagement of IFN-γ in intestinal inflammation. 49 They explained that telomere dysfunction of YAP1 up-regulates pro-IL-18. In the gut, the microbiome-activated caspase-1 cleaves pro-IL-18 into mature IL-18, subsequently recruiting the IFN-γ-secreting T cells and causes inflammation.
These findings motivated us to explore the role of YAP1 in tumor aggressiveness and immune evasion in breast invasive carcinoma and metastatic breast cancer with respect to IL-18. We unveil an important link between IL-18 and tumor-derived enhanced YAP1, which leads to a gene expression profile toward tumor promotion profiling.
It has also been shown the role of IL-18 as an inducer in the production of IFNG which ultimately affects oncogenesis. Even though substantial progress has been made in treating early stage and locally advanced breast cancers, the prognosis for patients suffering from the metastatic disease remains low. Despite noteworthy advances in the treatment of malignant growth, the metastatic type of the illness remains profoundly deadly, with a 5-year generally endurance pace of just around 20%. 50 About 70% of patients with metastatic sicknesses ineluctably become impervious to treatment. 51 Advances in disease immunotherapy with resistant checkpoint blockers have indicated that bridling the power of the body's invulnerable framework can be a useful technique to battle metastatic malignancy.     (Table 3)  ( Figure 4B). But in IFNG, there is a trend toward shallow deletion versus gains ( Figure 4C). Most of these breast cancer tissues exhibited increased DNA copy numbers, reflecting its consistent increased mRNA expression. In addition, the overall oncoprint of YAP1, IL-18

| MATERIALS AND METHODS
and IFNG gene expressions in Breast Invasive Carcinoma (PanCancer Atlas) cohort showed that mRNA expression was up-regulated by 3.02, 2.82, and 3.22% (YAP1, IL18 and IFNG) in all patients, respectively ( Figure 4D). Almost half of the parts of the population (2.72%) have been seen YAP1 down regulation. Therefore, our data support that YAP1 was increased in breast cancer ( Figure 2B), which might mainly originate from its increased DNA copy number and almost unchanged IL-18.  Figure 2B showed an increased YAP1 DNA copy number and almost unchanged IL18 DNA copy number results in metastasis.

| CONCLUSION
In conclusion, the present data analysis suggests that IL-18 might influence YAP1 in Breast oncogenesis through the production of

CONFLICT OF INTEREST
The authors declare there is no conflict of interest.

AUTHOR CONTRIBUTIONS
A.R., and L.S.S. conceived and designed the study. A.R. acquired the data. A.R. performed the analysis and interpreted the data. A.R. and L.
S.S. wrote the article. All the authors reviewed the article and gave final approval of the version to submit for publication.

DATA AVAILABILITY STATEMENT
Details of the data and analysis used in this study are available on reasonable request from the corresponding author.

ETHICAL STATEMENT
Not applicable.