beta-TrCP inhibition reduces prostate cancer cell growth via upregulation of the aryl hydrocarbon receptor

Abstract

Background: Prostate cancer is a common and heterogeneous disease, where androgen receptor (AR) signaling plays a pivotal role in development and progression. The initial treatment for advanced prostate cancer is suppression of androgen signaling. Later on, essentially all patients develop an androgen independent stage which does not respond to anti hormonal treatment. Thus, alternative strategies targeting novel molecular mechanisms are required. beta-TrCP is an E3 ligase that targets various substrates essential for many aspects of tumorigenesis.

Methodology/principal findings: Here we show that beta-TrCP depletion suppresses prostate cancer and identify a relevant growth control mechanism. shRNA targeted against beta-TrCP reduced prostate cancer cell growth and cooperated with androgen ablation in vitro and in vivo. We found that beta-TrCP inhibition leads to upregulation of the aryl hydrocarbon receptor (AhR) mediating the therapeutic effect. This phenomenon could be ligand independent, as the AhR ligand 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) did not alter prostate cancer cell growth. We detected high AhR expression and activation in basal cells and atrophic epithelial cells of human cancer bearing prostates. AhR expression and activation is also significantly higher in tumor cells compared to benign glandular epithelium.

Conclusions/significance: Together these observations suggest that AhR activation may be a cancer counteracting mechanism in the prostate. We maintain that combining beta-TrCP inhibition with androgen ablation could benefit advanced prostate cancer patients.

Figure 1. NF-κB activation correlates with prostate cancer patients' outcome.

Prostate cancer sections were immunostained using p65 antibodies. A. Representative photomicrographs of a primary tumor (left) and lymph node metastasis (right) from the same patient. B. Percent of negative, positive and strongly positive cases spotted on a tissue microarray from the indicated Gleason scores (p = 0.0014, Fisher's exact test). C. primary prostate tumors (n = 131) were immunostained for p65 and assessed for NF-κB activity. Kaplan-Meier plot revealed a correlation between NF-κB status and the risk for disease recurrence. Blue, strongly stained samples; Red, weakly stained samples; Green, negative stained samples (p = 0.02).

Figure 2. β-TrCP inhibition reduces prostate cancer cells growth.

LNCaP cells were infected with a lentiviral vector bearing an inducible doxycycline dependent β-TrCP shRNA. A. Cells were treated for 72 hours with 1 µg/ml doxycycline and RNA levels of β-TrCP1 and β-TrCP2 were measured using qRT PCR. B. Western blot analysis of protein extracts from cells treated with TNF, doxycycline or MG-132 were immunoblotted with either IκB or tubulin. C. Representative photomicrograph of cells treated as indicated and stained with hematoxylin and eosin. D. XTT assay used to quantify cell growth at different time points. DOX, doxycycline; NT, no treatment; FCS, conditioned medium containing fetal calf serum; CSS, conditioned medium containing androgen depleted serum (charcoal stripped serum). Error bars, SD. * Significantly different from non treated cells and from each other, p-value<0.01, t-test.

Figure 3. β-TrCP inhibition cooperates with androgen ablation treatment in vivo.

LNCaP cells bearing a tetracycline induced β-TrCP shRNA construct were injected subcutaneously to immunosuppressed Rag1 ^−/− mice. Mice (n≥4 in each group) were either untreated (NT), treated with tetracycline in their drinking water (Tet), physically castrated (Cast) or both (Cast+Tet) for 30 days. Tumor volumes were measured weekly. (A). qRT PCR for both β-TrCP isoforms was performed on RNA extracted from the tumors harvested at day 30. (B) Tumor growth kinetics. Tissue sections were stained for BrdU (C) or activated caspase 3 (D) and the proliferation and apoptosis scores, respectively, were determined for each tumor. Shown are mean ± standard deviation (A) or ±S.E.M (B, C and D).* p-value<0.05, ** p-value = 0.0002, t-test; p-value in C refers to t-test.

Figure 4. Aryl hydrocarbon receptor (AhR) expression is increased after androgen ablation and β-TrCP treatments.

A. LAPC4 cells infected with inducible β-TrCP shRNA were treated for 72 hours with the indicated treatment, subjected to RNA extraction and cDNA microarray analysis (Affymetrix). After data normalization, gene expression profiles were compared between treatment and the untreated control samples. A. Heat map dendrogram showing the ten most highly up (red) and down (green) regulated genes due to the combined treatment. Fold change refers to the combined treatment probes values relative to control. Expression of β-TrCP isoforms is presented below. B. LAPC4 cells infected with the same lentiviral vector and treated as indicated were subjected to RNA extraction and qRT PCR analysis with the listed primers. CSS, charcoal stripped serum; DOX, doxycycline; Error bars, SD.

Figure 5. AhR mediates the β-TrCP inhibition phenotype.

LNCaP cells infected with an inducible lentiviral vector for shAhR alone (A) or together with shβ-TrCP (B) were treated for 72 hours with the indicated treatments and cell growth was measured by the XTT assay. C. qRT PCR confirmed β-TrCP and AhR efficient knock down. D. Western blots showing β-catenin stabilization after β-TrCP inhibition and confirming AhR protein level elevation or knock down due to relevant shRNAs. DOX, doxycycline; FCS, fetal calf serum; CSS, charcoal stripped serum. Error bars, SD.

Figure 6. AhR is upregulated in malignant prostate cells.

Prostate sections were immunostained with AhR antibody. Photomicrographs show strong AhR expression in basal cells (arrows in A) and proliferative inflammatory atrophy (B). C. Higher AhR expression is noted in malignant glands (T) compared with normal glands (N). D. Normal and malignant glands were scored using a 0–3 scale. Mean values ±S.E.M. are shown (p-value<0.001, Mann-Whitney test).