**5. Mechanism of action of HE4 in angiogenesis**

Angiogenetic function of HE4 is promoted by epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and insulin. EGF & VEGF works through nuclear translocation, while insulin works through nucleolar translocation. EGF, VEGF and Insulin along with their receptors promotes tumour growth and proliferation in ovarian cancer [25–27]. VEGF is essential for hypoxia-inducible factor mediated neo-vascularisation and it is regulated by the hypoxia-inducible factor (HIF) family [28].

HE4 leads to the activation of protein STAT3, which gets phosphorylated by the receptor associated with Janus Kinases (JAK). It promotes translocation in the nucleus. Here it acts as a transcription activator.

The growth and proliferation of tumour cells leads to local hypoxia and inflammation leading to the activation of STAT3 to produce factors that promotes angiogenesis. VEGF is a potent proangiogenic factor that helps endothelial cells to induce angiogenesis. The signal from VEGF stimulates STAT3 that is responsible for endothelial cell proliferation. It also induces metaststic activity of tumour cells by regulating the transcription of the targeted genes.

Furthermore, STAT3 signalling promotes the up-regulation of pro-angiogenic STAT3 target genes IL8 and HIF1A in immune cells, ovarian cancer cells, and

endothelial cells. Moreover, HE4 promotes increase in tube formation in an *in vitro* model of angiogenesis, which is also dependent upon STAT3 signalling.

Firstly, the rapid proliferation of tumour cells leads to local hypoxia and inflammation, which activate STAT3 in tumour cells to produce pro-angiogenic factor. Further, VEGF (vascular endothelial growth factor) is a potent pro- angiogenic factor which promotes endothelial cell angiogenesis. The VEGF signal activates STAT3 which subsequently promotes endothelial cell proliferation and migration by regulating the transcription of the targeted genes.

Furthermore, STAT3 signalling promotes the up-regulation of pro-angiogenic STAT3 target genes IL8 and HIF1A in immune cells, ovarian cancer cells, and endothelial cells. Moreover, HE4 promotes increase in tube formation in an *in vitro* model of angiogenesis, which is also dependent upon STAT3 signalling.

Clinically, the positive correlation has been seen in between the serum levels of HE4 and IL8 in ovarian cancer patients. HE4 has been shown to be associated with microvascular density ovarian cancer tissue. HE4 is also shown to be inversely correlated with the amount of cytotoxic T cell infiltration. These phenomena suggest that HE4 may cause deregulated vascular proliferation and it suppresses T cell trafficking in tumour tissues [29].

HE4 has potential to alter the signalling pathways to modify the expression of related gene in the tumour micro-environment. Thus it affects angiogenesis and immunogenic responses in especially in ovarian cancer [29].

Tumour angiogenesis is also regulated by programmed cell death-1 (PD-1) that suppresses the anti-tumour function of CD8 + T cells [30].

The tumour vasculature is also regulated by the cytokines secreted by immune cells, and an interlinked activities have been studied between angiogenesis and immune suppression [31]. Human epididymis protein 4 (HE4) also functions as antiproteases [5, 6]. It inhibits the cytotoxic activities of mononuclear cells in the tumour micro-environment of ovarian cancer cells [7, 32].

HE4 promotes oncogenesis in ovarian cancer not only by promoting cell proliferation, metastasis, and chemo resistance, but it also by altering the tumour microenvironment. Because of being secretory protein, HE4 can function as intracellularly or by autocrine or paracrine mechanisms [1].

The angiogenesis mediated by immune cells is regulated through the activation of STAT3, which is mediated by HE4. STAT3 is responsible for immune suppression solid tumours [33–35]. The inhibitor T cell receptor ligand PD-L1 is also associated with tumour angiogenesis. It is regulated by HIF1α (Hypoxia-induciblefactor1-alpha) through transcription [36]. HIF1α binds to the HE4 gene promoter to up-regulate its transcription [37]. IL8 is a potent pro-angiogenic factor. Its expression is one of the poor prognostic factors of high-grade serous ovarian cancer [38]. HIF1α has also shown to promote angiogenesis and alters the metabolic environment in cancer [39, 40].
