LASS2 mediates Nrf2-driven progestin resistance in endometrial cancer

Abstract

Progestin administration serves as the optimal conservative treatment method for women with endometrial cancer or precancer lesions who want to preserve fertility. However, there are still at least 30% of patients in which progestin resistance occurs. LASS2 (Ceramide Synthase 2) has been reported to be involved in chemotherapy resistance, whether it also plays a role in progestin resistance is not clear. Here, we explored the detailed mechanism by which Nrf2/LASS2 contributes to progestin resistance and disease progression.

Methods: IHC assays were performed to estimate the expression pattern of Nrf2 and LASS2. Moreover, it bears three antioxidant response elements (ARE) in the promoter region of LASS2 gene, therefore, Luciferase assays were performed to determine if Nrf2 regulates LASS2 by binding with these ARE sequence. Western Blot assays were used to determine the expression of Nrf2 and LASS2 protein among various endometrial cell lines. Relative mRNA expression levels were detected by RT-PCR. Cellular growth was monitored with CCK-8 tests. Apoptosis was determined with Annexin V-PI staining and flow cytometry analysis. siRNA knockdown was performed to investigate the effects of Nrf2 on cell proliferation.

Result: Nrf2/LASS2 is highly expressed in endometrial cancer tissue, as compared to expression levels in normal endometrial tissue. Proliferation assays demonstrated that overexpression of Nrf2/LASS2 resulted in progestin resistance. Conversely, knockdown of LASS2 increased apoptosis and decreased cell viability. In addition, metformin overcame progestin resistance by down-regulating Nrf2/LASS2 expression.

Conclusion: Our findings provide new insight into the mechanism of progestin resistance in type I endometrial cancer. Nrf2/LASS2 may not only be a possible marker for predicting the prognosis of endometrial cancer but also serve as a potential therapeutic target.

Keywords: LASS2; Nrf2; Type I endometrial cancer; progestin resistance; proliferation.

Figure 1

Nrf2 regulates LASS2 and its functional effects on endometrial cancer cells. Dual luciferase assays to examine if Nrf2 drives LASS2 expression by binding with its AREs (A, B). Western blot was performed to determine the effect of t-BHQ on Nrf2 and LASS2 expression in protein levels and proliferation activity was evaluated by CCK8 assay (C, D). The apoptosis effect after knocking down LASS2 was monitored by FCM (E, F).

Figure 2

Nrf2 and LASS2 are positively correlated in endometrial cancers development. The expression of Nrf2 and LASS2 in benign and endometrial cancer tissues was assessed using IHC staining. Representative images were captured at 200 × magnification (A). Nrf2/LASS2 IHC mean of IOD in endometrial lesion tissues are presented (B, C). The expression of Nrf2 and LASS2 in benign and endometrial cancer cells was assessed using western blot (D) along with protein grayscale analysis (E). Analysis of the TCGA database displaying the relationship between Nrf2 and LASS2 (F).

Figure 3

Exogenous estrogen increase the expression of Nrf2/LASS2 which results in a decrease in long-term survival. RT-qPCR was used to detect mRNA levels of Nrf2 and LASS2 after estrogen stimulation (A), Western blot and proliferation analysis showing the effect of exogenous estrogen on Nrf2 and LASS2 expression (B, C). Analysis of the TCGA database showing high expression of Nrf2 correlates with a decrease in overall survival of endometrial cancer patients (D).

Figure 4

Nrf2 and LASS2 regulates progestin sensitivity in endometrial cancer cells. Relative cell viability was measured using CCK-8. Results demonstrate that Nrf2 and LASS2 expression have an impact on progestin resistance. Overexpression of Nrf2 or LASS2 results in increased viability (A, B) while knockdown of Nrf2 or LASS2 results in decreased cell viability (C, D). Western blots was performed to determine the efficiency of gene overexpression and knockdown.

Figure 5

Progestin withdrawal resulted in on-going Nrf2/Lass2 decline in protein level and attenuated proliferative activity. After 1 uM MPA treatment for the indicated 24 h, 48 h, and withdraw for 24 h after a 48 h treatment, followed by MPA withdrawl, the Nrf2 and LASS2 protein expression was determined by Western blot (A), the cellular growth was detected by CCK8 assay (B).

Figure 6

Nrf2/LASS2 mediate metformin-enhanced progestin sensitivity. Western blot analysis was used to detect the changes of Nrf2 and LASS2 protein levels after metformin treatment (A). CCK-8 was used to measure the relative cell viability after LASS2 knockdown (B) or LASS2 overexpression (C) cells. Colony formation assays shows the effects of exogenous drug on colony formation (D). Quantification of colony formation assays (E).

Figure 7

Proposed signaling pathway showing Nrf2/LASS2 regulation of the progression of endometrial cancer and progestin response. There is a feedback loop between Nrf2 signaling pathways and high levels of estrogen. Decreased Nrf2 contributes to enhanced progestin sensitivity, type I endometrial cancer patients with higher Nrf2 expression are at greater risk for disease progression.