Differential transformation capacity of Src family kinases during the initiation of prostate cancer

Abstract

Src family kinases (SFKs) are pleiotropic activators that are responsible for integrating signal transduction for multiple receptors that regulate cellular proliferation, invasion, and metastasis in a variety of human cancers. Independent groups have identified increased expression of individual SFK members during prostate cancer progression, raising the question of whether SFKs display functional equivalence. Here, we show that Src kinase, followed by Fyn kinase and then Lyn kinase, exhibit ranked tumorigenic potential during both paracrine-induced and cell-autonomous-initiated prostate cancer. This quantitative variation in transformation potential appears to be regulated in part by posttranslational palmitoylation. Our data indicate that development of inhibitors against specific SFK members could provide unique targeted therapeutic strategies.

Fig. 1.

Selective loss of SFKs differentially inhibit paracrine FGF10-induced PIN and carcinoma. (A) Schematic of prostate regeneration assay. Reconstituted prostate tissues are generated from a recombination of prostate epithelial cells with GFP(control)/FGF10-UGSM under subrenal capsule. (B) Paracrine FGF10-induced multifocal prostate adenocarcinoma shows elevated expression of activated Src kinase. Western and IHC analysis of pSrc(Y416), which cross-reacts with analogous sites in SFK members, in regenerated tissue derived from GFP or FGF10-UGSM. (Scale bar, 100 μm.) (C and D) Histological analysis of regenerated tissues by H&E and IHC for basal CK5 and luminal CK8. Regenerated tissues were derived from primary prostate cells of wild type, Src^−/−Fyn^+/−, Fyn^−/−, and Lyn^−/− combined with GFP-UGSM or FGF10-UGSM. Inserts provide high magnification to highlight cytokeratin expression. (Scale bar, 100 μm.)

Fig. 2.

Selective loss of SFKs led to a diminution of epithelial AR in response to paracrine FGF10 IHC analysis of AR and cyclin D1 expression in the regenerated tissue derived from primary prostate cells of wild type, Src^−/−Fyn^+/−, Fyn^−/−, and Lyn^−/− combined with FGF10-UGSM. (Scale bar, 100 μm.)

Fig. 3.

Overexpression of dominant negative Src kinase mutant inhibits paracrine FGF10-induced prostate adenocarcinoma. H&E staining, fluorescent microscopy, and IHC analysis shows histology, Src(Y529F/K298M)-infected RFP⁺ tubules, and expression of Src, AR, and cyclin D1 in regenerated tissues derived from primary prostate cells transduced with vector or Src(Y529F/K298M) and combined with FGF10-UGSM.

Fig. 4.

Ectopic expression of constitutively active Src family kinases in primary prostate cells indicates the hierarchical role of SFKs in the initiation of prostate cancer. (A) Schematic of prostate epithelial cell procurement, lentiviral infection (with dual-promoter vector encoding activated SFKs and the fluorescent marker RFP) and implantation to induce prostate carcinoma under subrenal capsule. The expression of SFK genes are driven by the ubiquitin promoter, whereas RFP is driven by the CMV promoter. (B) H&E staining, RFP signal, and IHC staining of CK5(red)/CK8(green) and AR in regenerated tissue derived from primary prostate cells infected with mAKT (control), Src(Y529F), Fyn (Y528F), and Lyn(Y508F). Inserts provide high magnification to highlight cytokeratin expression. (Scale bar, 100 μm.)

Fig. 5.

Alteration of palmitoylation sites modulate oncogenic potential of constitutively active Src and Fyn kinases in prostate cancer. (A) Schematic of SFKs mutations at palmitoylation sites. The serine 3 and 6 sites of Src(Y529F), and the cysteine 3 and 6 sites of Fyn(Y528F) were mutated to cysteine and serine, respectively. Src(Y529F/S3C/S6C) gains two palmitoylation sites while Fyn(Y529F/C3S/C6S) loses two palmitoylation sites. (B) Regenerated prostate grafts were derived from 2 × 10⁵ of prostate cells infected with Src(Y529F), Src(Y529F/S3C/S6C), Fyn(Y528F) or Fyn(Y528F/C3S/C6S). (C) H&E staining, RFP signal, and IHC staining of CK5(red)/CK8(green), E-cad(red)/Vim(green), Src kinase, phospho-Src (Y416), and phosphotyrosine in regenerated tissues derived from primary prostate cells infected with Src(Y529F), Src(Y529F/S3C/S6C), Fyn(Y528F), and Fyn(Y528F/C3S/C6S). Inserts provide high magnification to highlight cytokeratin expression. (Scale bar, 100 μm.)