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.

Src family kinases (SFKs) are a group of nonreceptor tyrosine kinases composed of nine highly homologous members with four conserved protein domains (1). All SFK members have an SH4 domain that mediates membrane association via myristoylation and, depending on the SFK, palmitoylation, as well as SH3 and SH2 domains that mediate inter- and intramolecular interactions, and finally the SH1 kinase domain (1). SFKs represent central convergence points for multiple receptors and cell-autonomous signaling pathways that mediate enhanced cellular proliferation, cell migration, and metastatic potential in cancer progression (1, 2). Drug resistance and failure to efficiently inhibit SFKs have spurred the development of a new generation of SRC inhibitors that are currently in clinical trials. These drugs represent a prospectively efficacious therapeutic strategy against numerous solid malignancies and second-line treatment of leukemia (3). However, due to the high homology of SFK members and kinase domains conserved in numerous receptor tyrosine kinases, these inhibitors targeting Src kinase and BCR/ABL also inhibit other SFK members and/or several receptor tyrosine kinases (3). Off-target effects by these drugs can impair normal tissue function, leading to clinically adverse symptoms including diarrhea, rash, and cardiac toxicity (4, 5).

Despite structural similarities, individual SFK members have specific cellular functions in normal development. Genetic knockout of Src, Lyn, or Fyn kinases in mice and derivative cells, respectively, exhibit defects in the development of bone, peripheral B cells, or T-cell receptor signaling (6–8). The specificity of individual SFK members may rely on their preferential association with cell surface receptors. For example, c-Src, but not Fyn or Lyn, is associated with α_IIbβ₃ integrin in blood platelet signal cascades (9). In contrast, Lyn and Fyn, but not Src, are preferentially recruited to the Fc receptor γ-chain and mediate platelet glycoprotein VI receptor signal transduction (10). In human glioblastoma cells, cooperation of integrin α_vβ₃ with platelet-derived growth factor receptor is dependent upon Lyn, but not Fyn, to regulate cell migration (11).

Previous studies from our laboratory have identified that enhanced expression of wild-type Src and androgen receptor (AR) in naive murine prostate cells results in poorly differentiated adenocarcinoma. This indicates that, whereas rarely mutated in human prostate cancer, Src kinase can still fulfill a functional role in prostate cancer initiation and progression (12, 13). This study, in light of the diverse cellular functions exhibited by SFK members, raises the question of whether individual SFKs are functionally equivalent with respect to tumorigenesis.

To investigate potential quantitative variation in transformation of prostate epithelium by SFK members, we used an in vivo prostate regeneration system that allows investigation of prostate transformation by both paracrine and cell-autonomous oncogenic stimuli (14, 15). Using this system, we investigated the differential functions of individual SFKs in mediating both paracrine FGF10-induced and cell-autonomous transformation of prostate epithelium. We demonstrate that individual SFK members are differentially used during FGF10-induced prostate cancer. Epithelial deficiency of Src kinase blocks FGF10-induced tumorigenesis and diminishes the heightened expression of epithelial AR normally associated with paracrine FGF10 signaling, whereas knockout of Fyn kinase partially inhibits transformation, and loss of Lyn kinase had no effect. We further demonstrate that SFKs have distinct roles in cell-autonomous initiation of prostate cancer. Ectopic expression of constitutively activated Src, Fyn, and Lyn kinases exhibit differential capacities for transformation of prostate epithelium. Src kinase presents the strongest oncogenic phenotype, followed by Fyn and then Lyn. Palmitoylation plays an essential role in mediating the distinct functions of Src and Fyn kinases with respect to prostate tumorigenesis. Gain of palmitoylation in Src kinase inhibited tumorigenesis induced by constitutively active Src kinase, whereas loss of palmitoylation of Fyn, but not Lyn, kinase accelerated tumorigenesis. These data collectively demonstrate that SFK members exhibit distinct intracellular functions and differential response to paracrine signals in the initiation of prostate cancer.

Results

Selective Loss of SFKs Inhibits Paracrine FGF10-Induced Prostatic Intraepithelial Neoplasia (PIN) and Carcinoma.

Aberrant paracrine signaling from the tumor microenvironment can act as a driving factor in tumorigenesis (16). FGF/fibroblast growth factor receptor (FGFR) paracrine signaling is one of many important pathways in the initiation of numerous cancers (17, 18). We have previously shown that chronic exposure to paracrine FGF10 leads to murine PIN (mPIN) and adenocarcinoma (14) with lesions exhibiting enhanced levels of phosphorylated SFK proteins (Fig. 1 A and B). Further, Western analysis of primary murine prostate tissue confirmed endogenous expression of SFK members Src, Fyn, and Lyn (Fig. S1A). This evidence, combined with studies reporting that Src kinase can mediate FGF signaling (2, 19), indicates functional utilization of SFK members in FGF10-induced transformation.

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.)

To assess differential utilization of SFK members during paracrine-induced prostate adenocarcinoma, we used mice bearing targeted knockouts of Src, Fyn, or Lyn kinase. Prostate epithelium from Src^−/−Fyn^+/−, Fyn^+/−, Fyn^−/−, or Lyn^−/− knockout mice and wild-type (WT) littermates were combined with FGF10- or GFP-transduced urogenital sinus mesenchymal cells (FGF10-UGSM or GFP-UGSM) (Fig. 1A). In a normal prostate regeneration system, UGSM provides an inductive environment for the regeneration of prostate tissue (20). Src^−/−Fyn^+/− mice were used because Src^−/−Fyn^−/− mice are embryonic lethal. Similar regenerative capacity and transformative response to FGF10 in WT controls suggests that genetic heterogeneity present in the different backgrounds does not affect these processes (Fig. S2). Further, the histology of regenerated tissue from Fyn^+/− with FGF10-UGSM or GFP-UGSM was similar to its WT counterpart (Fig. S2), indicating that Fyn haploinsufficiency does not affect transformation. Regenerated tissues from knockout mice combined with GFP-UGSM displayed normal prostate tubules containing CK8⁺ luminal cells and CK5⁺ basal cells and exhibited typical AR expression patterns (Fig. 1 C and D and Fig. S3). This suggests that Src, Fyn, and Lyn kinases are individually dispensable for regeneration of prostate glandular tissue.

Regenerated tissue from WT epithelia combined with FGF10-UGSM exhibited well-differentiated prostate adenocarcinoma, characterized by expansion of the CK8⁺ luminal population with few CK5⁺ basal cells (Fig.1 C and D). Similar to WT epithelium, tissues regenerated from Lyn^−/− epithelium combined with FGF10-UGSM displayed neoplastic growth (Fig. 1 C and D). Tissues from Fyn^−/− epithelial cells with FGF10-UGSM primarily exhibited mPIN lesions, characterized by an expansion of the CK8⁺ luminal population with maintenance of CK5⁺ cells (Fig. 1 C and D). In striking contrast, regenerated tissues from Src^−/−Fyn^+/− epithelium combined with FGF10-UGSM presented normal histology, indicated by normal glandular structures with CK8⁺ luminal and CK5⁺ basal epithelial layers (Fig. 1 C and D). Collectively, our results indicate that the oncogenic effects of FGF10 are largely mediated by Src and Fyn kinases in prostatic epithelium.

Selective Loss of SFKs Leads to a Diminution of Epithelial AR in Response to Paracrine FGF10.

Our previous study identified that the expression of epithelial AR increases in response to paracrine FGF10 signaling (14). To investigate modulation of AR expression in the context of paracrine FGF10 and selective loss of SFK members, we examined the expression of AR in regenerated tissues from SFK knockout tissue by immunohistochemistry (IHC).

Expression of AR in grafts derived from Src^−/−Fyn^+/−, Fyn^−/−, or Lyn^−/− epithelial cells with control UGSM was similar to grafts derived from WT littermates (Fig. S3). Selective loss of Lyn did not alter the expression pattern of AR and cyclin D1 in FGF10 grafts compared with WT prostate epithelia, whereas expression of AR and cyclin D1 was decreased to a lesser extent in tissues regenerated from Fyn^−/− epithelium (Fig. 2). In contrast, the epithelial expression of AR and cyclin D1 was down-regulated in tissues regenerated from Src^−/−Fyn^+/− epithelium combined with FGF10-UGSM cells compared with WT prostate epithelia. Collectively, the data indicate that loss of Src kinase, and to a certain extent loss of Fyn but not Lyn kinase, modulate expression of AR and cyclin D1 in response to paracrine FGF10.

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.)

Inhibition of Src Family Kinase Signaling by a Dominant Negative Src Kinase Mutant Attenuates FGF10-Induced Adenocarcinoma.

To support that Src family kinases mediate FGF10 signaling, we blocked SFK signaling by ectopic expression of Src(Y529F/K298M), an open conformation, kinase dead mutant of Src kinase (21). Dissociated prostate epithelial cells were transduced with either control vector or Src(Y529F/K298M) and combined with FGF10-UGSM cells. Both control and Src(Y529F/K298M) vectors contained an RFP fluorescent reporter driven by a separate CMV promoter. Tubules infected with control vector exhibited FGF10-induced adenocarcinoma (Fig. 3), with transformed tubules presenting increased AR expression. In contrast, tubules infected with dominant negative Src kinase were phenotypically normal and expressed low amounts of AR compared with neighboring RFP negative tubules (Fig. 3). As the dominant negative Src kinase could inhibit signaling through multiple SFK members, these data do not indicate hierarchical significance. However, they do strongly support a role for SFK signaling, mediating transformation and increased epithelial AR expression in response to chronic FGF10 signaling.

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.

Constitutively Active SFK Members Exhibit Differential Oncogenic Potential in Primary Prostate Cells.

Our results indicate that SFK members are not functionally equivalent in the context of paracrine-induced carcinoma; therefore, we asked whether this pattern was conserved in cell-autonomous transformation by SFK members. Independent laboratories have reported increased expression and activation of wild-type Src, Fyn, and Lyn with prostate cancer progression (22–24). We generated lentivirus bearing constitutively active Src(Y529F), Fyn(Y528F) or Lyn(Y508F) kinase with an RFP reporter (Fig. 4A) and confirmed expression by Western analysis (Fig. S1B). Although rarely observed in human cancers, constitutively active mutants phenocopy the synergy of c-Src and AR in prostate cancer and chronic SFK activation by signal transduction pathways (13).

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.)

To assess differential cell-autonomous transformation in primary cells, dissociated prostate epithelial cells were transduced with Src(Y529F), Fyn(Y528F), or Lyn(Y508F) kinase and combined with WT UGSM. Src(Y529F) tumors lacked glandular structure and were predominantly CK8⁺ luminal cells, characteristic of poorly differentiated invasive adenocarcinoma (Fig. 4B). Tubules overexpressing Fyn(Y528F) exhibited mPIN lesions with stratified layers of CK8⁺ luminal cells (Fig. 4B). In contrast, overexpression of Lyn(Y508F) resulted in phenotypically normal regeneration (Fig. 4B). Collectively, these in vivo results clearly demonstrate that cell-autonomous expression of constitutively active SFKs in naive adult prostate epithelium results in dramatically different phenotypes.

Alteration of Palmitoylation Sites Change Oncogenic Potential of Constitutively Active Src and Fyn Kinases in Prostate Cancer.

To investigate potential mechanisms for the observed differences in transformation between SFK members, we asked whether alteration of the palmitoylation status of SFK members modulates transformation capacity. Segregation of SFK members into lipid rafts by palmitoylation could further enhance preferential interactions with receptors and determine functional specificity (25, 26). To assess the role of palmitoylation of SFKs in determining oncogenic potential, wild-type and constitutively active Src and Fyn kinases, were respectively mutated at predicted palmitoylation sites (Fig. 5A) (25). We transduced Src^−/−Yes^−/−Fyn^−/− (SYF) fibroblasts with control or palmitoylation mutant Src and Fyn kinases and assessed in vitro colony formation in soft agar. Compared with controls, the Src(S3C/S6C) or Src(Y529F/S3C/S6C) palmitoylation mutants exhibited reduced colony formation and attenuated Src activation, whereas Fyn(C3S/C6S) or Fyn(Y528F/C3S/C6S) palmitoylation mutants exhibited dramatically increased colony formation or size of colony (Fig. S4). In addition, gain of palmitoylation sites in Src(S3C/S6C) or Src(Y529F/S3C/S6C) mutants attenuated expression of phospho-Src kinase (Fig. S4).

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.)

We then assessed transformation activity of Src palmitoylation mutants in the prostate regeneration assay. Src(Y529F) grafts displayed solid tumors without glandular structure (Fig. 5B) and exhibited primarily CK8⁺ but not CK5⁺ cells, both E-cadherin and vimentin expression, elevated phospho-Src(Y416), and phosphorylated tyrosine levels (Fig. 5C). In contrast, Src(Y529F/S3C/S6C)-infected tubules were predominantly normal with a few displaying low-grade hyperplasia. These tubules exhibited normal CK8 and CK5 patterns, expressed E-cadherin but not vimentin with low levels of phospho-Src(Y416) and phosphotyrosine. Additionally, both Src(Y529F/S3C/S6C) and Src(Y529F) regenerations displayed similar levels of total Src kinase (Fig. 5C). Finally, Src(Y529F) grafts also exhibited increased expression of phospho-ERK and phospho-FAK, but not Cbp and phospho-AR, compared with Src(Y529F/S3C/S6C) tissue (Fig. S5).

We next examined whether loss of predicted palmitoylation sites in Fyn kinase would likewise alter prostate transformation efficiency in vivo. In contrast to the mPIN lesions induced by Fyn(Y528F), lesions induced by Fyn(Y529F/C3S/C6S) presented as poorly differentiated invasive carcinoma and resembled transformation by Src(Y529F) (Fig. 5 B and C). The transformed tissues exhibited CK8⁺ but not CK5⁺ cells, vimentin but not E-cadherin expression, and highly elevated levels of pSrc(Y416) and phosphotyrosine (Fig. 5C). Fyn expression was assessed using a Src kinase antibody that exhibits cross-reactivity for other SFK members. The total Fyn expression was elevated in Fyn(Y529F/C3S/C6S)-transformed tissues compared with Fyn(Y529F) (Fig. 5C). In addition to changing how Fyn is trafficked within the cell, Fyn palmitoylation mutants could also exhibit higher stability, leading to more efficient expression (27, 28). Additionally, the expression of phospho-FAK was increased in Fyn(Y529F/C3S/C6S)-transformed tissue, but not the expression of Cbp, phospho-ERK, and phospho-AR (Fig. S5). Finally, expression of Lyn(Y508F) loss-of-palmitoylation mutants resulted in phenotypically normal regenerations (Fig. S6). Collectively, our studies suggest that palmitoylation modification of the SH4 domain modulates tumorigenic potential of constitutively active Src and Fyn kinases by regulating downstream signaling.

Discussion

Despite separate lines of evidence that indicate Src, Fyn, and Lyn kinases are each up-regulated in prostate cancer (22–24), our findings indicate that (i) individual SFK members differentially mediate paracrine FGF10 signal transduction and transformation and (ii) exhibit differential capacity for cell-autonomous transformation. SFKs have been considered as potential drug targets in prostate cancer. Dasatinib (Sprycel; Bristol Myers-Squibb), saracatinib (formerly AZD0530; AstraZeneca), and bosutinib (previously SKI-606; Wyeth) represent three inhibitors of Src kinase being used in the clinical trials (3). Dasatinib has high affinity for Src and BCR/ABL, but also targets other SFK members, c-KIT, PDGFR, and ephrin A2. Similarly, saracatinib can effectively inhibit Src and other SFK members with activity against ABL and activated mutant forms of EGFR, whereas bosutinib is a dual Src/ABL kinase inhibitor that also targets other SFK members without inhibition of KIT or PDGFR (3). Although these inhibitors exhibit clinical efficacy, reports have identified toxic effects, including centrosomal and mitotic spindle defects to normal cells, reduced tubular secretion of creatinine, and cardiac toxicity (4, 29, 30). Several adverse clinical symptoms such as renal failure, nausea, fatigue, lethargy, anorexia, proteinuria, vomiting, and diarrhea are also associated with treatment (3). Although the mechanisms leading to these adverse symptoms are unknown, given the functional differences of SFKs observed in our study, it becomes prudent to investigate whether selective, rather than broad, inhibition of SFKs could represent an effective treatment strategy and potentially reduce adverse effects.

The transformation capacity of SFK members is directly related to their differential localization within plasma membrane microdomains, which is determined in part by N-terminal lipid modification (25, 31). With respect to Src kinase, activity is seemingly dependent upon its distribution between plasma membrane microdomains that sequester inhibitory factors and substrate access outside of these domains (26). By enhancing the association of Src kinase with hydrophobic microdomains by artificial palmitoylation, its oncogenic activity is likely inhibited by endogenous regulatory mechanisms (26, 31). In contrast, loss of palmitoylation mutation in Fyn kinase results in gain of function that phenocopies activated Src kinase, likely due to some overlapping substrate specificities (32). This is also reflected in their differential responses to FGF and EGF signaling (33). In addition, modification of the N terminus of Src Family kinases, including palmitoylation and myristoylation, could alter their localization at cell membrane and subsequently influence protein expression and activity (27, 28). That mutation of palmitoylation sites in Lyn kinase does not increase transformation activity indicates that microdomain localization is not the sole determinant of activity and rather extends to substrate specificity as well. This notion is supported by studies identifying largely nonoverlapping signaling mechanisms (11) and trafficking patterns (34) between SFK members Src, Fyn, and Lyn. Finally, although our studies provide evidence that palmitoylation modification can modulate cell-autonomous transformation activity, it remains to be seen whether this plays a role in the human disease.

Our results support previous studies that FGF10-induced prostate adenocarcinoma exhibits elevated AR expression in epithelial cells (14). Over 80% of castration-resistant prostate cancer cases express high levels of AR and androgen-responsive genes (35). Our study suggests that specific SFK members are critical in mediating FGF10-induced transformation and the subsequent increase in AR expression, offering an in vivo mechanism linking FGF10 signal transduction and AR expression. Supporting a role for SFK members in modulating AR expression, a study by DaSilva et al. (36) identified that stabilization of AR by parathyroid hormone-related protein (PTHrP)/EGFR signaling is mediated by Src kinase. In our study, epithelial loss of Src kinase presented the greatest inhibitory effect on transformation and up-regulation of AR, indicating the greatest functional significance. Supporting this hypothesis, enhancement of FGF10/FGFR→Src kinase→AR signaling pathway by coexpression of wild-type Src kinase and AR in prostate epithelium results in a potent synergistic transformation phenotype (13). Collectively, these results imply that targeting this signaling pathway represents an important route for treating prostate tumorigenesis.

Materials and Methods

Plasmids.

Control FUCGW and FGF10-FUCGW vectors were prepared as described (14). The ORFs of murine Src and Fyn kinases were amplified by PCR from cDNA of mouse spleen or thymus using primer pairs of Src(F)-Gene and Src(R)-Gene, and Fyn(F)-Gene and Fyn(R)-Gene, respectively. The ORF of human Lyn kinase was PCR amplified from a plasmid generated as described (37). PCR products were cloned into the Xba1 and EcoR1 sites of the FUCRW lentivector, in which RFP is constitutively expressed by the CMV promoter (Fig. 2A). Constitutively active Src kinase was generated by site-directed mutagenesis with the QuikChange kit (Stratagene) using the primer pair Src(Y529F)-F/Src(Y529F)-R encoding for phenylalanine at residue 529. Constitutively active Fyn and Lyn kinase mutants were generated by PCR amplification using primer pairs Fyn(F)-Gene/Fyn(Y529F)-R and Lyn(F)-Gene/Lyn(Y508F)-R with substituted nucleotides encoding phenylalanine at residues 528 and 508, respectively. The tyrosine-to-phenylalanine mutation in SFK members allows adoption of an open conformation of the catalytic domain, leading to constitutive activation (38). Palmitoylation mutants of Src, Fyn, and Lyn kinases were generated by PCR amplification using primer pairs palm-Src-F/Src(R)-Gene or Src(Y529F)-R, palm-Fyn-F/Fyn(R)-Gene or Fyn(Y528F)-R, palm-lyn-F/Lyn(R)-Gene, or Lyn(Y508F)-R. Primer sequences are listed in Table S1 with underlined nucleotides indicating point mutations.

Mouse Strains.

Mouse strains used in this study include: (i) Fyn^+/−, Fyn^+/−Src^−/−, and wild-type littermates on a BL6/129S7 mixed genetic background and were maintained in Jonathan Cooper's laboratory; (ii) Fyn^−/− and wild-type littermates on a BL6/129S7 mixed genetic background and were purchased from Jackson Labs; and (iii) Lyn^−/− and wild-type littermates on a BL6 background and were maintained in Clifford Lowell's and Owen Witte's laboratories.

Regeneration and Transduction of Prostate Epithelium.

The regeneration process, lentivirus preparation, titering, and transduction of dissociated prostate cells were performed under University of California, Los Angeles safety regulations for lentivirus use as described previously (20). Lab animal housing, maintenance, and all surgical and experimental procedures were undertaken in compliance with the regulations of the Division of Laboratory Animal Medicine of the University of California, Los Angeles. Prostate regenerations were prepared as described (20). In brief, dissociated prostate cell suspensions were prepared from 6- to 10-wk-old male mice. A total of 1–2 × 10⁵ dissociated prostate cells were transduced with lentivirus carrying the gene of interest at a multiplicity of infection (MOI) of 50. Transduced cells were mixed with 1–2 × 10⁵ UGSM cells and suspended in collagen. Grafts were implanted under the kidney capsule in SCID mice and allowed to regenerate for 6–8 wk.

Immunohistochemistry and Western Analysis.

Following 1regeneration, hosts were killed and grafts were recovered via surgical resection of the kidney. Transilluminated and fluorescent images were taken using a dissecting microscope. Grafts were fixed in 10% buffered formalin overnight, embedded in paraffin, and sectioned at 4 μm. Sections were stained with hematoxylin and eosin (H&E) for representative histology. IHC stains were visualized using the EnVision+ system (Dako). Primary antibodies for Src kinase (1:250; Cell Signaling), phospho-Src family (Tyr416) (1:50; Cell Signaling), AR (1:200; Santa Cruz Biotechnology), phospho-AR (Ser213/210; 1:50; Imgenex), Cbp (ab14989; 1:200; Abcam), phospho-FAK (ab4803; 1:150; Abcam), and phospho-Erk (4376; 1:50; Cell Signaling) were used. For immunofluorescent analysis, sections were incubated with primary antibodies against vimentin (1:250; Abcam), E-cadherin (1:250; BD Transduction Laboratories), CK5 (Covance; 1:1,000), or CK8 (Covance; 1:1,000) and visualized by Alexa-594– or Alexa-488–conjugated secondary antibodies (Molecular Probes; 1:1,000). For biotinylated secondary antibodies, sections were incubated with FITC-conjugated streptavidin (Molecular Probes; 1:250). Sections were counterstained with DAPI (Vector Laboratories) and analyzed by fluorescent microscopy. Primary antibodies for phospho-Src family (Tyr416) (1:1,000; Cell Signaling), Erk2 (1:5,000; Santa Cruz Biotechnology) were used in Western analysis.