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B-cell-derived lymphotoxin promotes castration-resistant prostate cancer - PubMed

  • ️Fri Jan 01 2010

B-cell-derived lymphotoxin promotes castration-resistant prostate cancer

Massimo Ammirante et al. Nature. 2010.

Abstract

Prostate cancer (CaP) progresses from prostatic intraepithelial neoplasia through locally invasive adenocarcinoma to castration-resistant metastatic carcinoma. Although radical prostatectomy, radiation and androgen ablation are effective therapies for androgen-dependent CaP, metastatic castration-resistant CaP is a major complication with high mortality. Androgens stimulate growth and survival of prostate epithelium and early CaP. Although most patients initially respond to androgen ablation, many develop castration-resistant CaP within 12-18 months. Despite extensive studies, the mechanisms underlying the emergence of castration-resistant CaP remain poorly understood and their elucidation is critical for developing improved therapies. Curiously, castration-resistant CaP remains androgen-receptor dependent, and potent androgen-receptor antagonists induce tumour regression in castrated mice. The role of inflammation in castration-resistant CaP has not been addressed, although it was reported that intrinsic NF-kappaB activation supports its growth. Inflammation is a localized protective reaction to injury or infection, but it also has a pathogenic role in many diseases, including cancer. Whereas acute inflammation is critical for host defence, chronic inflammation contributes to tumorigenesis and metastatic progression. The inflammation-responsive IkappaB kinase (IKK)-beta and its target NF-kappaB have important tumour-promoting functions within malignant cells and inflammatory cells. The latter, including macrophages and lymphocytes, are important elements of the tumour microenvironment, but the mechanisms underlying their recruitment remain obscure, although they are thought to depend on chemokine and cytokine production. We found that CaP progression is associated with inflammatory infiltration and activation of IKK-alpha, which stimulates metastasis by an NF-kappaB-independent, cell autonomous mechanism. Here we show that androgen ablation causes infiltration of regressing androgen-dependent tumours with leukocytes, including B cells, in which IKK-beta activation results in production of cytokines that activate IKK-alpha and STAT3 in CaP cells to enhance hormone-free survival.

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Figures

Figure 1
Figure 1. Androgen ablation induces tumor inflammatory infiltration

Six week old FVB males (n=10) were inoculated with myc-CaP cells. When tumors reached 1000 mm3, mice were left untreated, castrated or sham operated. Tumors were collected when indicated for analysis. A. Paraffin-embedded tumor sections were TUNEL stained to determine apoptotic cell frequency (results are averages, n=3). B. Total RNA was isolated from tumor samples and expression of indicated cell marker mRNAs was quantitated and normalized to that of cyclophilin A (norm= normal, C1,2,3= mice analyzed 1,2,3 weeks after castration, sham= sham-operated). Results are averages ± s.d. (n=10). C. Frozen human prostate sections (normal tissue [n=3], prostatic hyperplasia [n=3], and malignant CaP with Gleason scores 6-8 [n=10]) were stained with CD4, CD8 and CD20 antibodies and DAPI and analyzed by immunofluorescent microscopy. The histogram denotes average frequencies of indicated cell types (n=3 per sample). P values were determined and are depicted as insignificant (ns), significant (*), very significant (**) or highly significant (***).

Figure 2
Figure 2. Role of B cells and IKKβin STAT3 activation and CR-CaP emergence

A. Myc-CaP tumors were established in WT mice reconstituted with BM from Rag1−/− males (n=10) or in Rag1−/− males. When tumors reached 1000 mm3, mice were castrated. Three days before castration, Rag1−/− mice (n=10 per group) received via the tail vein purified splenic B or T cells. Tumor volume was measured. Results are averages ± s.e.m.. P values were determined and are indicated as above. B. Tumors were removed from radiation chimeras reconstituted with IkkβF/F, IkkβΔ/Δ, or Rag1−/− BM, one week after castration. STAT3 phosphorylation was analyzed by immunohistochemistry. C. Tumor-bearing Rag1−/− males were injected with WT splenocytes (Rag1 + WT), or purified splenic B (Rag1 + B) or T (Rag1 + T) lymphocytes. One day later, mice were castrated and after one week, tumors were removed and analyzed for STAT3 phosphorylation.

Figure 3
Figure 3. Role of IKKα in emergence of CR-CaP

Tumor-bearing mice were castrated or sham operated as above. A. Tumors were analyzed one week later for nuclear IKKα by immunohistochemistry. B. Tumors removed at indicated times were divided into cytosolic (C) and nuclear (N) fractions and IKKα and histone H3 distribution was determined. C. Tumors were established using myc-CaP cells transduced with lentiviruses expressing scrambled siRNA (sc) or IKKα-specific siRNA. Mice were castrated as above and tumor volume was measured. Results are averages ± s.e.m. (n=10). P values were determined and are indicated as above. D. Tumors were established in lethally irradiated FVB males reconstituted with IkkβF/F or IkkβΔ/Δ BM or in Rag1−/− males reconstituted with either B or T cells. IkkβF/F and IkkβΔ/Δ chimeras were i.p. injected three times with poly(IC) (250 μg) prior to castration to delete IKKβ. One week after castration, tumor samples were analyzed for IKKα distribution by immunohistochemistry. Nuclear IKKα results in punctuate staining, while cytoplasmic IKKα results in diffuse staining.

Figure 4
Figure 4. IKKβ-dependent lymphotoxin production by tumor-infiltrating B cells stimulates IKKα-dependent androgen-free survival

A. RNA from splenic B cells of IkkβF/F and IkkβΔ/Δ mice was analyzed for LTα and LTβ expression as above. Results are averages ± s.d. (n=3). B. Lethally irradiated FVB males were reconstituted with BM from B-Ltβ−/− or T-Ltβ−/− mice (n=6 per group). After 8 weeks, myc-CaP tumors were established, mice were castrated and tumor volume was measured as above. Results are averages ± s.e.m.. C. FVB mice (n=6 each group) bearing myc-CaP tumors were castrated and given hIgG or LTβR-Ig (100 μg) every 5 days, starting 4 days before castration. Tumor volume was measured as above. Results are averages ± s.e.m.. D. Tumors were established using myc-CaP cells transduced with lentiviruses expressing scrambled (sc) siRNA or LTβ-specific siRNA. Mice were castrated and tumor volume was measured. Results are averages ± s.e.m. (n=10). E. Myc-CaP cells (previously infected with lentiviruses expressing scrambled or IKKα siRNAs) were plated at 40% confluency. After 6 hrs, the cells were cultured with or without flutamide (10 μM) in the absence or presence of LTα2β1, and cell number was determined. F. Myc-CaP cells were plated at 60% confluency. After 12 hrs, cells were stimulated for 1 hr with LTα2β1, collected, divided into cytosolic (C) and nuclear (N) fractions and IKKα and histone H3 distribution was determined. In A, B, C, D and E, P values were determined and are indicated as above.

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