Ovarian cancer is the seventh most common malignant tumor worldwide. In 2012, there were an estimated 238,719 cases of ovarian cancer, showing an age-standardized rate of 6.1/100,000 . The American Cancer Society predicted that in 2020, there will be approximately 21,750 new ovarian cancer cases in the United States, and 13,9400 women will die from this disease. Because of its occult onset and innocuous symptoms, most patients with ovarian cancer are diagnosed at an advanced stage. Despite the development of new anti-tumor drugs and improvements in surgical treatment, the survival rates for ovarian cancer decline dramatically from 92% for patients with stage I disease to 17–28% for those with the advanced stages of this disease (stages III–IV) ; most patients at an advanced stage eventually relapse and show chemotherapeutic resistance. Although the serum biomarker CA125 is widely used in clinical practice for the diagnosis and differentiation of ovarian cancer, population-based screening of serum CA125 and use of the risk for ovarian cancer algorithm (ROCA) did not result in significant mortality reduction and is thus, ineffective . Thus, the mechanism underlying the oncogenesis of EOC must be determined, and the identification of tumor biomarkers is necessary to facilitate the early diagnosis and targeted therapy or prevention of this disease.
As a new tumor biomarker, HE4 has gained attention in recent years. However, most studies focused on its clinical application with regard to the early and differential diagnosis of EOC, and the prediction of relapse, prognosis, chemotherapeutic resistance, as well as other clinical aspects of EOC [12, 35]; few studies have been performed to determine the mechanism underlying its role in ovarian cancer. This may be because HE4 is not considered as a therapeutic target, as its roles in EOC tumorigenesis and progression remain controversial. In 2011, Gao et al.  reported that the overexpression of HE4 markedly promoted ovarian cancer cell apoptosis and adhesion, and that HE4 may inhibit ovarian cancer cell proliferation, migration, and invasiveness, as well as xenograft tumor formation in vivo; thus, they concluded that HE4 may play a protective role in the progression of EOC. In 2014, Kong et al.  performed in vitro studies that showed that this protective influence may be attributed to the regulation of the MAPK and PI3K/AKT pathways. In contrast, other researchers noted that high HE4 expression promotes cell migration, adhesion, proliferation, and spreading, which are associated with effects on the EGFR-MAPK signaling pathway [18, 36]. Additionally, HE4 contains a fucosylated modification (Lewis y antigen) , the overexpression of which can promote HE4-mediated invasion and metastasis in ovarian cancer cells . Overexpression of Lewis y antigen enhanced the tyrosine phosphorylation of EGFR and HER/neu, improving cell proliferation via the PI3K/AKT and Raf/MEK/MAPK pathways . Based on these results, Lewis y antigen and HE4 may affect similar signaling pathways to promote tumor growth and malignancy . HE4 overexpression promotes ovarian cancer cell xenograft tumor growth in vivo, which can be suppressed by an antisense target of HE4. HE4 interacts with tumor microenvironment constituents (EGFR, IGF1R, insulin) and the transcription factor HIF1α, supporting that HE4 is related to growth factor signaling and the MAPK/ERK pathway . Annexin A2 was identified as a robust interacting partner of HE4 by mass spectrometry and co-immunoprecipitation, and the HE4-Annexin A2 complex was found to promote the invasion and migration abilities of ovarian cancer cells in vitro and tumor distant metastasis of the lung in vivo. Downregulation of HE4 decreases the expression of MKNK2 and LAMB2, which are associated with MAPK signaling pathways and focal adhesion .
In recent years, an increasing number of studies have shown that HE4 promotes the proliferation, adhesion, invasion, migration, and chemoresistance in ovarian cancer cells [20–26, 42–47]. HE4 overexpression in, or recombinant HE4 treatment of, EOC cells resulted in the upregulation of many transcripts coding for extracellular matrix proteins, including LAMC2, LAMB3, SERPINB2, and GREM1; moreover, in cells overexpressing HE4 or those exposed to recombinant HE4 in the culture medium, the protein levels of LAMC2 and LAMB3 were observed to continuously increase. In the presence of fibronectin, focal adhesions were elevated in cells treated with recombinant HE4 .
Ovarian cancer participates in the evasion of immunosurveillance and orchestrates a suppressive immune microenvironment. A series of studies by James et al. [23, 44, 45] showed that upon exposure of purified human peripheral blood mononuclear cells to HE4, osteopontin (OPN) and DUSP6 were the most downregulated and upregulated genes, respectively. The proliferation of human ovarian carcinoma cells in conditioned media from HE4-exposed peripheral blood mononuclear cells was enhanced, whereas this effect was attenuated by adding recombinant OPN or OPN-inducible cytokines (interleukin-12 and interferon-γ). HE4 can compromise both OPN-mediated T cell activation  and the activity of cytotoxic CD8+/CD56+ cells by upregulating self-produced DUSP6 , thus promoting the tumorigenesis of ovarian cancer [23, 44, 45, 48]. Other researchers revealed that HE4 promotes the carcinogenesis of ovarian cancer by combining with histone deacetylase 3 to activate the PI3K/AKT pathway , and that HE4 knockdown suppresses the invasive cell growth and malignant progress of ovarian cancer by inhibiting the JAK/STAT3 pathway . Few studies have examined the role of HE4 in the chemoresistance of ovarian cancer. Overexpression of HE4 promotes the collateral resistance of ovarian cancer cells to cisplatin and paclitaxel, and downregulation of HE4 partially reverses the resistance of ovarian cancer cells to multiple chemotherapeutic agents; HE4-mediated chemoresistance may be related to various factors, including deregulation of MAPK signaling (EGR1 and p38 inhibition) and alterations of tubulin levels or stability. Recombinant HE4 may upregulate the levels of α-tubulin, β-tubulin, and microtubule-associated protein tau [41, 43]. Similarly, in vitro, HE4 represses apoptosis induced by carboplatin, and recombinant HE4 results in increased Bcl-2 expression and decreased Bax (Bcl-2-associated X protein) expression in carboplatin-treated ovarian cancer cells, reducing the Bax/Bcl-2 ratio. In addition, HE4 suppresses EGR1 expression, which may contribute to the overall reduction of the expression of pro-apoptotic factors that lead to EOC chemoresistance . HE4 can enhance the regulation of, and is positively correlated with, DUSP6 expression. DUSP6 deactivates extracellular signal-regulated kinase (ERK), and inhibition of DUSP6 can alter the gene expression of ERK pathway response genes (EGR1 and c-JUN) and sensitize ovarian cancer cells to chemotherapeutic agents (paclitaxel or carboplatin) [23, 48]. Resensitization of ovarian cancer cells to cisplatin and paclitaxel caused by HE4 knockdown occurs because of corresponding decreases in the activities of ERK and AKT pathway-associated genes during gene knockout, and the activation of these pathways inhibits apoptotic signaling in tumor cells .