The crosstalk between β-catenin signaling and type I, type II and type III interferons in lung cancer cells

Abstract

Background/objective: IFNs induce potent antiviral and antitumor activities. β-catenin pathway is a surviving pathway adapted by carcinogenetic mechanisms of various cancers. Crosstalk between these pathways has not been well described in lung cancer cells.

Methods: Lung cancer cell lines, A549 and Calu-3, were used in this study. β-catenin protein levels and signaling activities were tested by flow cytometry and luciferase assay. Cell proliferation was measured by counting viable cells under microscope, and apoptosis by TUNEL assay and caspase 3 activation. DKK1 and GSK3β levels were tested by flow cytometry. Secreted DKK1 was measured by ELISA. αDKK1 , FLUD and S3I were to inhibit DKK1, STAT1 and STAT3 activities, respectively.

Results: All of IFNα, IFNγ and IFNλ1 suppressed β-catenin signaling in A549 and Calu-3 cells, where IFNγ was the strongest (P<0.05). They inhibited cellular proliferation and promoted apoptosis. IFNγ gave greater induction ability compared to IFNα and IFNλ1 (P<0.05). All tested IFNs promoted DKK1 activation but not GSK3β in A549 and Calu-3 cells. IFNs activated STAT1 and STAT3. But only STAT3 was vital for IFN-mediated DKK1 activation and apoptosis. Plus, DKK1 antagonist abrogated IFN-mediated apoptosis. The degree of STAT3 activation was corresponding to the level of apoptosis induced by different IFNs (P<0.05).

Conclusions: In lung cancer cells, all three types of IFNs can induce apoptosis via suppressing β-catenin signaling by a STAT3- and DKK1-dependent manner. This findings demonstrate a link between IFNs and β-catenin signaling, which may possess potentials on the development of novel therapeutic measures against lung cancer.

Keywords: crosstalk; interferon; lung cancer; β-catenin.

Figure 1

IFNs down-regulate β-catenin activation and signaling activity. Lung cancer cell lines, A549 (A) and Calu-3 (C), were left untreated or treated with IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ1 (100 ng/ml) for 24 hrs before transfection with TOPflash luciferase and Renilla luciferase constructs. After 4 hrs, the cells were cultured with or without initial treatment of different IFNs. Dual luciferase activity was measured 24 hrs later. Data shown is normalized to Renilla activity. A549 (B) and Calu-3 (D) were treated with or without IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ1 (100 ng/ml) for 48 hrs and expression of hypophosphorylated/active β-catenin level was measured by flow cytometry. Data represent a minimum of three experiments and asterisks denote P<0.05 in comparison to untreated samples.

Figure 2

Cell proliferation and apoptosis can be regulated by IFNs via β-catenin signaling in HCC. A549 (A) and Calu-3 (D) cells were untreated or treated with IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ1 (100 ng/ml) for 72 hours. Viable cells were then counted. The abscissa represents different types of stimulation. The ordinate represents percentage of viable cells relative to mock cells. Apoptosis in A549 (B, C) and Calu-3 (E, F) was measured by TUNEL assay. The ordinate represents fold increase of fluorescence intensity relative to untreated cells. Data represent a minimum of three experiments and asterisks denote P<0.05 in comparison to untreated samples.

Figure 3

Apoptosis can be regulated by IFNs via β-catenin signaling in HCC. A549 (A) and Calu-3 (B) cells were untreated (dashed line) or treated with IFNα (100 ng/ml, solid line), IFNγ (100 ng/ml, long dashed line) or IFNλ1 (100 ng/ml, dotted line) for 72 hours. Active caspase 3 was measured by flow cytometry. Data represent a minimum of three experiments and asterisks denote P<0.05 in comparison to untreated samples.

Figure 4

IFNs up-regulate DKK1 expression but show no effects on GSK3β. A549 and Calu-3 cells were treated with or without IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ1 (100 ng/ml), and levels of DKK1 were measured by ELISA (A, C) at 48 hours post-treatment. Activated GSK3β level was evaluated by flow cytometry in A549 and Calu-3 cells at 48 hours upon different IFN stimulation (B, D). Data represent a minimum of three experiments and asterisks denote P<0.05 in comparison to untreated samples.

Figure 5

STAT activation induced by IFNs in HCC. A549 cells were untreated or treated with IFNα, IFNγ or IFNλ1 for 0.5 hour in the absence (black bars) or presence of STAT1 inhibitor (FLUD, grey bars) or STAT3 inhibitor (S3I, open bars). Active STAT1 (A) and STAT3 (B) levels were measured by flow cytometry. Data represent a minimum of three independent experiments. Asterisks denote P<0.05 in comparison to untreated samples.

Figure 6

IFN-induced stimulation of DKK1 is STAT3-dependent. A549 (A) and Calu-3 (B) cells were untreated or treated with FLUD, S3I, or αDKK1 alone or combined with IFNα, IFNγ or IFNλ1, respectively for 48 hours. DKK1 levels were evaluated by ELISA. Data represent a minimum of three independent experiments. Asterisks denote P<0.05 in comparison to untreated samples.

Figure 7

IFN-induced apoptosis in lung cancer cells is DKK1 and STAT3-dependent. A549 (A) and Calu-3 (B) cells were left untreated or treated with FLUD, S3I, or αDKK1 alone or in combination with IFNα, IFNγ or IFNλ1, respectively for 72 hrs. The levels of induced apoptosis were measured by TUNEL assay. Data represent a minimum of three independent experiments. Asterisks denote P<0.05 in comparison to untreated samples.