Role of Human Epididymis Protein 4 in Tumour Angiogenesis

Abstract

HE4 is a secretory protein. It is expressed in reproductive tract and respiratory epithelium in normal individuals. Serum level of HE4 is raised in various solid cancers that give us an advantage to use it as a diagnostic and prognostic biomarker. It is an established biomarker of epithelial ovarian cancer [EOC]. It has also shown the significance in various other malignancies like cancer of endometrium, cervix, lung and breast. Studies show HE4 as an independent prognostic biomarker in non-small cell lung carcinoma. Its raised values in cancer signify its role in oncogenesis. HE4 promotes angiogenesis via STAT3 signalling pathway. In this paper we have tried to illustrate about human epididymis protein 4 and its role in tumour angiogenesis.

1. Introduction

Ovarian cancer is one of the most common gynaecologic malignancies that have the highest mortality rate. Ovarian cancer is the third most common [after cervical and corpus uteri] gynaecological malignancy worldwide and it is the most deadly gynaecological malignancy [1]. The disease produces few and nonspecific symptoms early in the course of illness. Consequently about 70% of the cases are diagnosed in the advanced stage [stage 3 and 4] with a 5 year survival of less than 30%. The disease has a good prognosis if diagnosed in the early stages, with a 5 year survival rate in stage 1 disease being 83.4–89.6% [2].

Therefore it is imperative to diagnose the disease early to improve prognosis and reduce mortality.

Diagnosis is usually based on clinical examination followed by transvaginal ultrasonography. Both these methods suffer from low specificity and subjectivity and are thus not recommended for screening.

The differentiation between benign and malignant ovarian disease is often only possible after histological examination of the resected ovarian tissue. It subjects the women to unnecessary surgical procedures which could be avoided if reliable, accurate, noninvasive tests were to become available as a majority of ovarian masses in both premenopausal and postmenopausal women are benign.

This is followed by measurement of serum biomarker levels the most common being CA125, and HE4. Though CA125 is an established biomarker, it has a low specificity and can be elevated in other benign gynaecological conditions and malignancies. 40–50% patients with epithelial ovarian cancer [EOC’s] have normal CA125 levels [3].

HE4 is the only other biomarker approved by FDA for monitoring response to treatment and recurrence of disease in EOC. HE4 is a secretory protein of the whey acidic protein [WAP] family. The expression of HE4 is restricted to reproductive tract and respiratory epithelium in normal individuals. HE4 levels are over expressed in EOC tissues and serves as a sensitive and specific serum marker for diagnosis, prognosis and disease recurrence. It is also less likely to be elevated in benign conditions. HE4 levels may also be elevated in other malignancies such as endometrial cancer and pulmonary neoplasms and has been proposed as a biomarker for these conditions [4].

2. Expression of HE4 in tumours

HE4 is a secretory glycoprotein. It is over-expressed in ovarian carcinomas especially in serous and endometriod variants. It is also called whey-acidic-protein that belongs to WAP domain family. WAP is a four-disulfide core domain protein 2 [WFDC2]. It was initially described to have tissue specific expression in epididymis [5].

Available literature revealed that HE4 is expressed in female reproductive tract, breast tissue, kidney, regions of the respiratory tract and nasopharynx [6, 7, 8]. Moreover, it is also expressed in normal human trachea, salivary glands, lung, prostate, pituitary gland, thyroid, and kidney [8]. It’s an independent prognostic marker in non-small cell lung cancer [NSCLC] [9].

3. HE4 as a biomarker in ovarian cancer

HE4 is a complementary biomarker to CA125 (Carbohydrates Antigen125). HE4 has potential to complement or even is a better alternative to carbohydrate antigen 125 (CA125) [10, 11, 12]. Currently CA125 is being used as a biomarker for the diagnosis and therapeutic monitoring in ovarian cancer. CA125 is raised in only 50% of stage I epithelial ovarian cancers, and only about 80% in all epithelial ovarian cancers [13]. So for all practical purposes, CA125 cannot be used as an independent biomarkerfor the diagnosis of EOC, while HE4 can be used as a stand-alone biomarker, in both diagnosis and prognosis of EOC and endometrial cancers [14, 15]. It has also been approved by the United States of America (USA), Food and Drug Administration (FDA) as a biomarker to monitor patients with epithelial ovarian cancer [16].

Studies show that serum level of HE4 and CA125 together can be used as an indicator of prognosis in ovarian cancer. The two together has shown positive results, sensitivity of 76.4% and specificity of 95% [17]. Individually, HE4 is a well known biomarker for the diagnosis and therapeutic monitoring for ovarian cancer.

4. Role of HE4 in the pathogenesis of ovarian cancer

HE4 is also involved in the pathogenesis of ovarian cancer. It aids in cellular proliferation, tumour growth, metastatic ability, chemoresistance and suppresses cytotoxic effect of mononuclear cells on ovarian carcinoma cells [18]. An inverse relationship between serum HE4 levels and CD8+ T cells in EOC has also been noted [19, 20].

Angiogenesis is one of the hallmarks of cancer and is necessary for bringing oxygen and nutrients to tumour cells and removal of waste products. It aids in tumour growth and invasion [21].

Hypoxia inducible factor 1 alpha (HIF1a) is a transcription factor that is involved in adaptation of cells to a hypoxic environment through its pro-angiogenic actions.

Interleukin 8 (IL-8) is also an important pro angiogenic factor produced by infiltrating macrophages in the tumour. Both of these factors are up-regulated by STAT3.

Recent studies have shown that HE4 promotes angiogenesis and is dependent on intact STAT3 signalling for its action. The addition of STAT3 inhibitors ablated elevated HIF1a levels in an in vitro experiment involving ovarian cancer cell lines and also blocked tube formation in human umbilical vein endothelial cell [19].

Several patients of ovarian cancer experience a chemo resistant recurrence within 2 years after first line therapy (cytoreduction surgery and platinum based chemotherapeutics) [18].

The diverse effects of HE4 that aid in tumour progression makes it an attractive therapeutic target for EOC and may serve as a effective treatment option for recurrent chemoresistant cases.

HE4 is produced as a, 13 kDa protein and converted to a, 25 kDa secreted glycosylated protein. HE4 is a highly over-expressed in epithelial ovarian cancer (EOC) [15, 22, 23, 24] compared to normal ovarian epithelium. US-FDA has approved HE4 as a biomarker for the diagnosis of ovarian cancer especially in the presence of an adnexal mass as part of the Risk of Ovarian Malignancy Algorithm (ROMA) [2].

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, 26, 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, 34, 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].

6. Role of HE4 in tumorigenesis

HE4 promotes tumorigenesis through its pro-angiogenic effects. Various studies have shown the role of HE4 in tumour angiogenesis. HE4 modulates angiogenesis by regulating the expression of different genes in multiple cell types of the tumour environment [19]. HE4 activates protein kinase B (AKT) which plays a role in tumour angiogenesis [41, 42]. James et al. have shown that HE4 activates signal transducer and activator of transcription 3 (STAT3) signalling pathway which up-regulates the pro-angiogenic genes interleukin 8 (IL8), hypoxia inducible factor 1A (HIF1A) in ovarian cancer cells, endothelial cells, and immune cells [19, 42, 43]. Levels of both HE4 and the pro-angiogenic factors were found elevated in ovarian cancer tissue in comparison to adjacent normal ovarian tissue [19, 42]. IL8 and HIF1A are potent angiogenic factors that promote tumour vessel formation and tumour growth [42]. IL8 also leads to persistent neutrophil recruitment in the tumour tissue which stimulates neoangiogenesis [44, 45]. HIF1A plays a role in hypoxic adaptation of cancer cells [46, 47]. STAT3 inhibitors block the effect of HE4- mediated tube formation of endothelium by suppressing STAT3 activation and down-regulating IL8 and HIF1A [19]. HE4 has been shown to be associated with increased levels of matrix metalloproteinases which are responsible for angiogenesis [48, 49]. Levels of HE4 and interleukin-1 alpha (IL1A) are directly proportional [50, 51]. IL1A promotes VEGF formation [52]. Annexin II (ANXA2) too is involved in angiogenesis and its gene expression has been increased by HE4 [53]. HE4 has also been found to promote tube formation in in-vitro model of angiogenesis [19]. Serum levels of HE4 positively correlated with the microvascular density in ovarian cancer tissue as reflected by the increased CD34+ areas in the tumour tissue. However, the new vessel formation is dysregulated [19]. These dysfunctional tumoral vasculature impair the movement of cytotoxic T cells along with other immune cells involved in host’s anti-tumour immune responses [54].

7. Prognostic and predictive value of HE4 in ovarian cancer

Presently, there is no predictive biomarker for the success of chemotherapy. Studies show that HE4 and CA 125 values become negative after fourth cycle chemotherapy if there is a good response. Rise in HE4 is seen earlier than CA 125. Long term progression-free survival is associated with the serum levels of biomarker lower than the mean value in the affected population at the time of diagnosis and the development of negativity of the marker after third cycle of chemotherapy. Monitoring of HE4 and Ca125 during chemotherapy especially after third cycle is recommended for their prognostic values [55].

8. Conclusion

HE4, a secretory glycoprotein is over-expressed in ovarian cancer most commonly in the serous and endometriod variants. It is a complementary biomarker to CA125. When HE is used in combination with CA125, the diagnostic and prognostic performance of ovarian cancer is increased significantly. HE4 is involved in oncogenesis by promoting cellular growth, proliferation, metastasis and chemoresistance in ovarian cancer. HIF1a and Interleukin 8 are associated pro-angiogenetic factors, which are up-regulated by STAT3 pathway. Angiogenetic action of HE4 is also associated with epidermal growth factor, vascular endothelial growth factor (VEGF) and insulin which promoted nuclear translocations. So, research on HE4 may decode some novel mechanisms of oncogenesis to provide alternative therapeutic options.

Conflict of interest

“The authors declare no conflict of interest”.