Inhibition of DUSP6 sensitizes ovarian cancer cells to chemotherapeutic agents via regulation of ERK signaling response genes

Abstract

Dual specificity phosphatase 6 (DUSP6) is a protein phosphatase that deactivates extracellular-signal-regulated kinase (ERK). Since the ovarian cancer biomarker human epididymis protein 4 (HE4) interacts with the ERK pathway, we sought to determine the relationship between DUSP6 and HE4 and elucidate DUSP6's role in epithelial ovarian cancer (EOC). Viability assays revealed a significant decrease in cell viability with pharmacological inhibition of DUSP6 using (E/Z)-BCI hydrochloride in ovarian cancer cells treated with carboplatin or paclitaxel, compared to treatment with either agent alone. Quantitative PCR was used to evaluate levels of ERK pathway response genes to BCI in combination with recombinant HE4 (rHE4), carboplatin, and paclitaxel. Expression of EGR1, a promoter of apoptosis, was higher in cells co-treated with BCI and paclitaxel or carboplatin than in cells treated with chemotherapeutic agents alone, while expression of the proto-oncogene c-JUN was decreased with co-treatment. The effect of BCI on the expression of these two genes opposed that of rHE4. Pathway focused quantitative PCR also revealed suppression of ERBB3 in cells co-treated with BCI plus carboplatin or paclitaxel. Finally, expression levels of DUSP6 in EOC tissue were evaluated by immunohistochemistry, revealing significantly increased levels of DUSP6 in serous EOC tissue compared to adjacent normal tissue. A positive correlation between HE4 and DUSP6 levels was determined by Spearman Rank correlation. In conclusion, DUSP6 inhibition sensitizes ovarian cancer cells to chemotherapeutic agents and alters gene expression of ERK response genes, suggesting that DUSP6 could plausibly function as a novel therapeutic target to reduce chemoresistance in EOC.

Gene Expression Profile of Active HE4 Stimulation in Epithelial Ovarian Cancer Cells: Microarray Study and Comprehensive Bioinformatics Analysis

“…In recent years, a growing number of investigations have gradually found that HE4 promotes cell proliferation, adhesion, invasion, migration, and chemoresistance in ovarian cancer [20][21][22][23][24][25][26][42][43][44][45][46][47]. It was found that HE4 overexpression or rHE4 treatment in EOC cells resulted in upregulation of many transcripts coding for extracellular matrix proteins, including LAMC2, LAMB3, SERPINB2 and GREM1; moreover, in cells overexpressing HE4 or exposed to rHE4 in culture medium, the protein levels of LAMC2 and LAMB3 were continously increased, and in the presence of bronectin, the focal adhesions were elevated in cells treated with rHE4 [22].…”

“…It was known that ovarian cancer participates in evading immunosurveillance and orchestrating a suppressive immune microenvironment, a series of studies by James NE, et al [23,44,45] found that, upon exposure of puri ed human peripheral blood mononuclear cells(PBMCs) to HE4, osteopontin (OPN) and DUSP6 appeared as the most inhibited and upregulated genes; the proliferation of human ovarian carcinoma cells in conditioned media from HE4-exposed PBMCs was enhanced, while the effect was attenuated by adding recombinant OPN or OPN-inducible cytokines (IL-12 and IFN-γ); HE4 can compromise both OPN-mediated T cell activation [44] and cytotoxic CD8 + /CD56 + cells through upregulation of self-produced DUSP6 [45], thus promoting the tumorigenesis of ovarian cancer [23,44,45,48]. Other researchers found that HE4 promotes carcinogenesis of ovarian cancer by combining with histone deacetylase 3 (HDAC3) to activate PI3K/AKT pathway [46], and that HE4 knockdown suppresses the invasive cell growth and malignant progress of ovarian cancer by inhibiting JAK/STAT3 pathway [24].…”

“…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, which reduces the ratio of Bax/Bcl-2; in addition, HE4 also suppresses EGR1 expression, which may contribute to the overall reduction of pro-apoptotic factors that lead to EOC chemoresistance [26]. HE4 can enhance the regulation of DUSP6 and they are positively correlated, DUSP6 deactivates extracellular-signal-regulated kinase (ERK), the inhibition of DUSP6 can alter gene expression of ERK pathway response genes (EGR1 and c-JUN) and sensitize ovarian cancer cells to chemotherapeutic agents(paclitaxel or carboplatin) [23,48]. The resensitization of ovarian cancer cells to cisplatin and paclitaxel caused by HE4 knockdown is due to the corresponding decreases of ERK and AKT during gene knockouts, and the activation of these pathways inhibits the apoptotic signal of tumor cells [21].…”

“…Furtherly, it was found in vitro that HE4 may regulate the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT signal transduction (PI3K/AKT) pathways to produce tumor-suppressing effect [18,19]. Recently, emerging studies have been carried out to investigate the association between HE4 and tumorigenesis as well as chemotherapeutic resistance in EOC, whereas the studies were inconclusive due to inconsistencies in results [20][21][22][23][24].…”

Gene expression profiles following active HE4 stimulation in epithelial ovarian cancer cells: microarray study and comprehensive bioinformatics analysis

“…Further, HE4 may regulate the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT signal transduction (PI3K/AKT) pathways to exert tumor-suppressive effects in vitro [18,19]. Recently, studies have been carried out to investigate the association between HE4 and tumorigenesis as well as chemotherapeutic resistance in EOC; however, these have shown inconsistent results [20][21][22][23][24].…”