Abstract
Prostate cancer (CaP) recurrence after androgen ablation therapy remains a significant cause of mortality in aging men. Malignant progression and metastasis are typically driven by genetic and epigenetic changes controlled by the androgen receptor (AR). However, evidence suggests that activated nonreceptor tyrosine kinases, including those of the Src family kinases (SFK), directly phosphorylate AR, thereby activating its transcriptional activity in the absence of serum androgen levels. To ascertain whether CaP progression and metastasis require SFK members, an autochthonous transgenic adenocarcinoma (AD) of the mouse prostate (TRAMP) model was crossed into Src-, Lyn- or Fyn-null backgrounds. Primary-site CaP formation was dependent on Src, to a lesser extent, Lyn, but not Fyn. Only Src−/−;TRAMP prostate tumors were marked by reactive stroma. SFK deficiency did not affect progression to neuroendocrine (NE) disease, although there were fewer new cancer cases initiating after 34 weeks in the SFK−/−;TRAMP mice compared with TRAMP controls. Of note, 15% to 21% of older (>33 weeks) Lyn- or Fyn-null TRAMP mice lacking primary-site tumors suffered from aggressive metastatic AD growths, compared with 3% of TRAMP mice. Taken with the data that TRAMP mice lacking Src or Lyn exhibited fewer macroscopic metastases compared with Fyn−/−;TRAMP and TRAMP controls, this suggests that SFK can either promote or suppress specific parameters of metastatic growth, possibly depending on cross-talk with primary tumors. These data identify critical, yet potentially opposing roles played by various SFKs in the initiation and metastatic potential of CaP using the TRAMP model.
Implications: Genetically defined mouse models indicate a critical role for Src tyrosine kinase in CaP initiation and metastatic progression. Mol Cancer Res; 12(10); 1470–9. ©2014 AACR.
Introduction
Prostate cancer (CaP) is an endocrine malignancy whose strict growth requirement for androgens such as dihydrotestosterone gave rise to so-called androgen-deprivation therapy (ADT), in which antiandrogens such as bicalutamide induce tumor regression by starving the androgen receptor (AR) of serum ligand levels. In the naïve, androgen-stimulated setting, AR functions as an androgen-dependent transcription factor that controls the expression of proliferation and survival genes in concert with scores of co-activator or -repressor proteins. ADT failure is marked by castration-recurrent (CR) CaP in the prostate and/or metastatic sites, especially draining lymph nodes and bones. Interestingly, CR-CaP lesions typically express wt-AR that is active in the absence of serum androgen levels, yet still responsive to very low, intracrine levels of androgens thought to be produced at the tumor site (1, 2).
Several mechanisms for AR activation in the CR setting have been described (2), including (i) overexpression of wt-AR, (ii) expression of AR mutants or splice variants (3, 4), (iii) alterations in AR coregulator expression (5), and (iv) AR modification such as by activated serine/threonine kinases (6, 7), and tyrosine kinases such as Ack1 (8) and Src family kinases (SFK; ref. 9). Progression to CR-CaP is marked by increased total protein phosphotyrosine (10) and relative Ack1 and SFK autophosphorylation/kinase levels (8, 9, 11), likely induced downstream of several receptor tyrosine kinases (RTK) and G-protein coupled receptors known to be activated during CaP progression. Src potentiates AR function in human CR-CaP cell lines (12, 13) and activated Src is sufficient to induce CaP formation in tissue recombination models involving AR overexpression (10, 14, 15). Indeed, androgen-independent AR transactivation function can be induced following phosphorylation on Y267 by Ack1 (8) or on Y534 by Src (9, 16). The growing appreciation for the role of SFK and Ack1 in AR-dependent CaP progression, as well the role played by SFK in metastatic invasiveness to and growth in the bone environment (14, 16–20), have served as the rationale for a growing number of clinical trials incorporating SFK and broad tyrosine kinase inhibitors in CR-CaP (21–23).
TRAMP (transgenic adenocarcinoma of mouse prostate) is an autochthonous model in which CaP adenocarcinoma (AD) is induced by the prostate epithelial cell–specific inactivation of p53 and Rb via the expression of SV40 T-antigen driven by a truncated probasin gene (Arr2bp) promoter (24). In this model, an AR-negative neuroendocrine (NE) cancer eventually takes over, seeding both primary prostate sites as well as multiple metastatic sites. We addressed whether CaP formation, progression to NE disease, and metastatic formation in this model would be affected by the loss of Src, Lyn, or Fyn, SFK members known to be expressed in human prostate epithelial cells (25). Our data indicate that CaP formation is suppressed by Src and to a lesser extent, Lyn, but not by Fyn, and that in mice forming CaP tumors, loss of the SFK did not prevent the onset of primary-site NE disease. Interestingly, whereas Src and Lyn seem to be required for NE metastasis, a small percentage of older Fyn- and Lyn-deficient mice lacking primary-site tumors exhibited highly aggressive metastatic AD. These data validate the concept of therapeutically targeting SFK to prevent initiation and progression of CaP disease.
Materials and Methods
Animals and genotyping
B6;129S7-Fyntm1Sor/J mice (stock number 002385), B6;129S4-Lyntm1Sor/J mice (stock number 003515), and B6;129S7-Srctm1Sor/J mice (stock number 002277) were purchased from The Jackson Laboratory. TRAMP mice were kindly obtained through the Mouse Tumor Model Resource, Roswell Park Cancer Institute (RPCI; Buffalo, NY). Male and female Fyn−/− and Lyn+/− mice were bred to homozygous TRAMP+/+ positive males and females on an FVB background to produce Fyn−/−;TRAMP+/wt and Lyn−/−;TRAMP+/wt males and females. Src+/− males and females were mated to homozygous TRAMP+/+ male and female mice on an FVB (Friend Virus B) background to produce Src−/−;TRAMP+/wt male and Src+/−;TRAMP+/wt female mice. TRAMPwt/wt littermates were used for negative controls. Src−/− female mice have impaired fertility (26), and thus, Src+/− were used as breeders. Mice that are Src−/− also exhibit decreased length of long bones leading to a noticeably smaller body size compared with that of heterozygous littermates, slower weight gain, delayed eye lid opening and incisors that fail to erupt (http://jaxmice.jax.org/strain/002277.html), requiring a soft food diet (catalog number S4798-TRAY, Bio Serv), and delayed weaning until 5 weeks of age. Src−/− mice exhibit an increased mortality rate in the first 3 to 5 weeks compared with Src+/− littermates.
Genotyping was performed using DNA extracted as described previously (27) from tails snips taken at weaning. Genotyping was performed by PCR (1 cycle 94°C for 3 minutes; 35 cycles, 94°C for 30 seconds; 54°C for 45 seconds; 72°C for 45 seconds); primers sets and products sizes for wild-type (WT) and mutant genomes are as follows: Fyn-WT, 415 bp; mutant, 190 bp. Lyn-WT, 340 bp; mutant, 280. Src-WT, 200 bp; mutant, 450 bp. Casein 500 bp. TRAMP 600 bp. PCR products were separated by gel electrophoresis on 1.5% agarose gels and visualized following staining with ethidium bromide.
Tumor palpation and tissue collection
Beginning at 8 weeks of age, all male mice were weighed, checked weekly for tumor or metastasis formation by palpation, and tumor volumes, measured by caliper, were calculated using the formula, 
, where L is the longest dimension and W is the width. Tumor volume was used as the endpoint unless health or distress issues required earlier euthanasia as recommended by veterinary staff. Animals were anesthetized with isoflurane and blood was collected as a terminal bleed via cardiac puncture. Tumors, metastases, and organs were removed postmortem, and were either used to produce lysates for immunoblotting or fixed in formalin for hematoxylin and eosin (H&E) and IHC analyses. All procedures were in accordance with approval by the RPCI Institutional Animal Care and Use Committee.
Immunohistochemistry and tissue imaging
Immunohistochemistry (IHC) was performed as described previously (28) using a purified mouse anti-SV40 Large T Antigen (Tag; BD Biosciences), polyclonal rabbit anti-synaptophysin (Invitrogen), and polyclonal rabbit anti-cytokeratin, wide spectrum screening (DAKO) Tissues were fixed in 10% neutral buffered formalin, placed in paraffin, and sectioned at 5 μm. Tissue sections were deparaffinized rehydrated with a graded alcohol series, and rinsed in distilled H2O. Antigen retrieval was performed with 1X citrate buffer (pH 6.0; Invitrogen). After washing, endogenous peroxidase activity was blocked with a 3% H2O2 in methanol solution and equilibrated with 1X Tris-PO4 buffer (10× solution 8.4 mmol/L Na2HPO4, 3.5 mmol/L KH2PO4, 10 mmol/L Tris, 120 mmol/L NaCl, 0.5% Tween-20, pH 7.8) followed by blocking in 1% BSA/Tris-PO4. Primary antibodies (Ab) were diluted in 1% BSA/Tris-PO4, placed on tissue sections and incubated overnight at room temperature in a humidified chamber. Slides were washed and the secondary Ab, rabbit anti-mouse or goat anti-rabbit (DAKO) was applied to each tissue section for 2 to 4 hours. Ab staining was visualized using a 1 mg/mL 3′3-diaminobenzidine chromogen (Sigma) and 1 μL/mL H2O2 solution. Slides were counterstained with Meyer hematoxylin, dehydrated, and coverslipped. Standard staining methods were used to produce H&E staining of all tissues. IHC slides were scanned into an Aperio Spectrum Digital Slide system, and images were viewed and captured using Aperio ImageScope software (Aperio Technologies, Inc.). Microscopic metastases were identified by a combination of analysis of H&E and SV40 Tag staining.
Immunoblotting and coimmunoprecipitation
Immunoblotting was performed as previously described (29) on dorso-lateral lobe RIPA buffer lysates using Ab specific for Fyn (FYN3; Santa Cruz Biotechnology), Lyn (#44; Santa Cruz Biotechnology), Src (Cell Signaling Technology), phosphoY416-Src (Cell Signaling Technology), and Gapdh (Santa Cruz Biotechnology). Coimmunoprecipitation (Co-IP) analyses were performed on dorsal-lateral prostate (DLP) RIPA lysates from 8-week-old mice with early palpable CaP tumors as follows: 500 mg total protein per sample was incubated for 8 hours at 4°C with 1 μg AR Ab (Santa Cruz Biotechnology; catalog #sc-816), then incubated overnight with A/G agarose beads (Santa Cruz Biotechnology). After three washes with PBS, the IPs were subjected to immunoblotting analysis with either AR Ab (Santa Cruz Biotechnology; cat. #sc-819) or anti-poY MAb-4G10 (Millipore; cat. #05-1050X). Digital imaging and signal quantification were performed on a Chemi-Genius2 Bio-Imager (Syngene) using GeneTools software.
Statistical analyses
Statistical analyses were performed by using the GraphPad Prism v5 (GraphPad Software). Differences in tumor incidence were assessed using the Fisher exact test.
Results
To assess the role of specific SFK members in CaP initiation, progression, and metastatic potential, TRAMP mice, carrying the SV40 Tag driven by the probasin Arr2bp promoter (30), were crossed onto Src-, Lyn-, or Fyn-null backgrounds. Src protein and activation levels increase throughout the initiation and progression phases of CaP-AD and -NE in the TRAMP model (12), suggesting that rising SFK levels might contribute to TRAMP oncogenesis. Deficiency of each of the SFK members was confirmed by immunoblots of prostate dorso-lateral lysates (vs. those from TRAMP mice) using Src-, Lyn-, and Fyn-specific monoclonal antibodies (Fig. 1A). There was no evidence that the loss of one specific SFK led to the compensatory overexpression of other SFKs. Similarly, relative levels of SFKpoY416, which recognizes a shared SFK autophosphorylation site and which correlates with SFK activity (31), were not grossly affected by the loss of Src, Lyn, or Fyn (Fig. 1A). Although there was some variation in the relative SFKpoY416 levels between individual SFK−/−;TRAMP mice, overall relative SFKpoY416 levels increased in all genotypes as the mice aged. This may reflect increasing activation of upstream RTKs during CaP progression. Thus, any suppression of tumor initiation in the SFK−/−;TRAMP mice relative to TRAMP controls would likely be due to the loss of Src, Lyn, or Fyn rather than the upregulation of activity by other SFK members.
Tumor formation in the TRAMP model in the absence of SFK. A, loss of Src, Lyn, or Fyn was confirmed by immunoblotting of dorso-lateral CaP lysates from either TRAMP control mice (left) or in SFK−/−;TRAMP mice (right), in comparison with relative levels of SrcpoY416 (correlating with overall SFK activation levels) or Gapdh (as a protein loading control); N.S., nonspecific. B, tumor formation versus age (in weeks) of the SFK−/−;TRAMP versus TRAMP controls based on the following numbers of mice studied and statistical power relative to TRAMP controls: Src−/−;TRAMP-36 (P = 0.001), Lyn−/−;TRAMP-60 (P = 0.143), Fyn−/−;TRAMP-72 (P = 0.735), TRAMP-10; +, censored. Note that while every TRAMP mouse (10/10) developed a tumor by 36 weeks of age (median 23 weeks), the Src−/−;TRAMP mice developed tumors more slowly (median 36 weeks) and less frequently (10 of 26 mice total). In addition, there is no significant difference in mice that develop tumors or live to 36 weeks without tumors: overall log-rank P = 0.09; Fyn−/−;TRAMP versus TRAMP, P = 0.578; Lyn−/−;TRAMP versus TRAMP, P = 0.38; Src−/−;TRAMP versus TRAMP, P = 0.172. C, smoothed hazards for TRAMP versus Lyn−/−;TRAMP mice showing decreased hazard for tumor incidence in older (>29 weeks) Lyn−/−;TRAMP mice. Shaded region reflects the difference incidence between genotype groups (blue, TRAMP and red, Lyn−/−;TRAMP) before 24 weeks and after 29 weeks. Permutation P values (calculated by log-rank test) are based on the area shaded in blue if the group identifiers are randomly re-sorted. D, left, DLP lobe lysates from 8-week-old SFK-null;TRAMP or TRAMP mice (3 mice/group) were immunoprecipitated using AR-specific Ab and then analyzed by immunoblotting for AR or total phosphotyrosine (poY). Right, the relative AR-poY from each group was quantified by densitometric analysis as described in Materials and Methods, using the mean value for parental TRAMP = 1; error bars, SE of 3 mice per group; *, P < 0.01 using the Student one-tailed t test.
The oncogenic progression of CaP in TRAMP mice is marked by the onset of AR-positive AD from 8 to 12 weeks after birth followed by the onset and eventual predominance of NE disease. In contrast, although limited developmental defects are detected in select SFK-deficient mice, such as osteopetrosis in Src-null mice (32), the loss of Src, Lyn, or Fyn alone is not sufficient to induce developmental or prooncogenic effects in the prostate, suggesting that the redundant expression of other SFK is compensatory for the lost function. To assess how the loss of specific SFK affected TRAMP initiation and progression, we identified cancer cases based on the appearance of palpable DLP tumors, which occurred in 48% of parental TRAMP mice by 20 weeks of age (Fig. 1B), in agreement with previous assessments (24). The kinetics of tumor initiation in Fyn−/−;TRAMP and Lyn−/−;TRAMP mice during weeks 10 to 28 was indistinguishable from that of parental TRAMP mice. The Fyn−/−;TRAMP mice continued to exhibit similar tumor initiation kinetics to that of TRAMP mice through 34 weeks, after which, there was a slight slowdown in tumor initiation rate in Fyn−/−;TRAMP mice, whereas there was a statistically significant slowing of the tumor initiation rate in Lyn−/−;TRAMP mice after week 30 compared with the parental TRAMP mice (Fig. 1C). In contrast, Src−/−;TRAMP mice showed much less tumor initiation starting at week 18 (P = 0.001). These data indicate that TRAMP-associated tumor initiation is dependent on Src, somewhat on Lyn but less so on Fyn. It should be noted that SFK losses did not alter the propensity of the CaP lesions to form in DLP lobes.
To address possible mechanisms for the relatively suppressed oncogenesis in Src−/−;TRAMP mice, we immunoprecipitated AR from DLP lysates of SFK-null;TRAMP or TRAMP mice (3 mice each) during early AD formation (8-week-old), immunoblotted these proteins using anti-phosphotyrosine antibody, and then quantified the relative AR-poY levels by densitometry. Early-onset lesions were chosen because, compared with NE lesion, which are typically AR-negative, they would more likely be driven by AR-mediated signaling. Figure 1D shows that the relative AR-poY levels in Src−/−;TRAMP AD lesions were significantly lower than those in parental TRAMP, Fyn−/−;TRAMP, or Lyn−/−;TRAMP AD lesions. This correlates with the decreased early tumor formation Src−/−;TRAMP mice (Fig. 1B), and suggests that Src is the most critical of the three SFK in activating AR function by direct phosphorylation.
The loss of Fyn, Lyn, or Src had different effects on the overall pathologic progression of AD or NE CaP disease compared with TRAMP controls based on, respectively, cytokeratin or synaptophysin expression (Table 1). For example, most AD-only tumors in Fyn−/−;TRAMP or Lyn−/−;TRAMP mice occurred early (weeks 21–28), although this was a little delayed compared with TRAMP controls, which had their highest incidence in 16- to 20-week-old mice. Src−/−;TRAMP mice displayed AD-only tumors even later, from weeks 25 to >32. Similar to TRAMP controls, AD/NE-combination tumors appeared in Fyn−/−;TRAMP throughout the 16 to 32 weeks period. In contrast, Lyn−/−;TRAMP and Src−/−;TRAMP showed much later development of AD/NE-combination tumors. Whereas more NE-only tumors were found in older TRAMP mice (25–32 weeks), there was a paradoxical early appearance of these tumors in 16- to 20-week-old SFK−/−;TRAMP mice, and these lesions were marked by reactive stroma, based on increased mesenchymal layers and cellular infiltrates (Fig. 2A). Later forming NE CaP tumors, based on a dearth of cytokeratin staining and an abundance of synaptophysin staining, could be found in all SFK−/−;TRAMP and TRAMP groups (Fig. 2B). As a control, we showed that the unaffected ventral prostate (VP) lobes of control C57BL/6 mice had abundant cytokeratin staining throughout the luminal cell layer, but only few synaptophysin-positive cells in the basal layer (inset, arrows). Taken together, these data indicate that the loss of specific SFK affected the initiation and progression of AD and NE disease in the TRAMP model.
Effect of SFK loss on TRAMP pathogenic progression. A, comparison of dorso-lateral CaP tumors in 20-week-old Src−/−; or Fyn−/−;TRAMP mice, showing extensive stromal formation in the absence of Src. B, ventral lobes from 16- to 25-week-old SFK−/−;TRAMP, TRAMP or C57BL/6 mice were analyzed by H&E staining or by IHC for SV40 Tag, high molecular weight cytokeratins (CK) or synaptophysin. Inset, ×60 magnification showing typical cell–cell CK staining in the luminal epithelium of normal VP lobes with rare peripheral synaptophysin-positive cells (arrows). C, NE LN-metastases in 19- to 25-week-old SFK−/−;TRAMP or TRAMP mice based on synaptophysin-positive, CK-negative staining.
Incidence of AD versus NE tumors in SFK−/−;TRAMP versus TRAMP mice
We then analyzed whether the loss of SFK affected the relative proliferation, cell death, and Tag expression rates in 20-week-old mice. Proliferation was measured by Ki67 staining whereas cell death was measured by TUNEL staining (Table 2). Although the Lyn−/−;TRAMP CaP proliferation rate was less than half that of the Fyn−/−;TRAMP and Src−/−;TRAMP tumors, it was comparable with that of TRAMP tumors. Cell death rates were statistically comparable between the genotypes. Finally, there was a slight decrease in the Tag positivity in Src−/−;TRAMP tumors, possibly owing to the aforementioned increase in reactive stroma.
Rates of proliferation, cell death, and T-antigen expressiona
To assess whether SFK play roles in general or organ-specific metastasis formation, 16- to 33-week-old TRAMP or SFK−/−;TRAMP mice bearing roughly equally sized primary tumors were examined postmortem for macroscopic metastases. Whereas Fyn−/−;TRAMP mice exhibited similar rates and organ distribution of metastases compared with parental TRAMP mice, Lyn−/−;TRAMP and Src−/−;TRAMP mice were relatively deficient in macroscopic metastases (Table 3; ref. 33). Indeed, most metastasis formation in all mouse crosses was detected in local draining pelvic lymph nodes, yet the Lyn−/−; TRAMP and Src−/−;TRAMP mice exhibited little or no macrometastasis formation in other peripheral organs. Similar results were found when micrometastases were analyzed (Table 4). The vast majority of these metastases were of NE origin (Fig. 2C), most likely arising from primary-site NE tumors. Taken together, these data indicate that NE macrometastasis formation is suppressed in the absence of Src and Lyn, but not in the absence of Fyn.
Incidence of macroscopic metastases in tumor-bearing micea
Incidence of microscopic metastases in tumor-bearing micea
We noted that between 35% and 65% of SFK−/−;TRAMP mice older than 16 weeks remained tumor free, as defined initially by palpation and then by postmortem pathologic confirmation, compared with 41% of TRAMP controls. However, whereas only 3% of tumor-free TRAMP mice exhibited metastases, 21% of Fyn−/−;TRAMP and 15% of Lyn−/−;TRAMP mice were found to have macrometastases postmortem; one 36-week-old Src−/−;TRAMP mouse was tumor free yet presented with a large pelvic lymph node mass (5 g), initially thought to be a palpable prostate tumor, plus renal metastases and abdominal ascites (Table 5). Except for the single Src−/−;TRAMP case, these mice were identified on the basis of a lack of palpable prostate tumors yet rapid onset of cachexia or even sudden death, suggesting an underlying aggressive disease. Unlike the typical NE metastases found in tumored mice (Fig. 2C), which were found mainly in draining lymph nodes, these lesions were distributed throughout multiple organs, and moreover, they displayed AD rather than NE markers (Fig. 3A and B). This suggests that Fyn and Lyn may suppress the early dissemination of AD progenitor cells, which might evolve into metastatic growths independent of primary CaP progression.
AD lung metastases in SFK−/−;TRAMP or TRAMP mice lacking primary-site tumors. A, lung and VP lobe pairs from individual >33-week-old SFK−/−;TRAMP or TRAMP mice stained as in Fig. 2B, showing either hyperplastic or high-grade prostatic intraepithelial lesions in the VP lobe simultaneous with AD lung metastases. Note that this Src−/−;TRAMP case had one small tumor each in the dorsal prostate (DP; arrow) and VP lobes and, thus, was not scored as prostate tumor-free in the Src−/−;TRAMP column in Table 5. All images are at ×5 magnification. B, cytokeratin and synaptophysin staining in examples of the metastases cited in Table 5, scored as the number of prostate tumor–free mice over total number of mice studied per genotype, with percentages in parentheses; *, see Src−/−;TRAMP case above.
Incidence and sites of macroscopic metastases in tumor-free micea
Discussion
An increasing number of studies demonstrate that CR-CaP, the lethal phenotype of CaP, is driven by the activation of AR in the absence of serum androgen levels. Data showing that AR can be activated by direct phosphorylation by Ack1 and SFK nonreceptor tyrosine kinases, whose activities increase during CaP progression, strongly suggest that this pathway may help drive CR-CaP progression. Indeed, activated versions of these kinases are sufficient to induce androgen-independent growth of CaP cell lines (8, 9), and activated Src plus AR overexpression can induce CaP in tissue recombination models using primary prostate epithelial cells (14, 15). A study by Cai and colleagues (14) ranked Src as most able to induce CaP initiation using this latter system, with Fyn and then Lyn ranking progressively less active.
This study is the first to address whether specific SFK members are required for CaP initiation, progression of NE tumors, and metastatic potential. In agreement with the tissue recombination studies, Src was found to be the most potent CaP inducer. This correlated with our finding that relative AR-poY was decreased only in early Src-null;TRAMP CaP lesions (8 weeks), likely to reflect AR-driven AD lesions. Moreover, our study indicates that Src and Lyn are equally critical to the formation of distal macrometastases in the TRAMP model. Importantly, the suppression of AD and NE formation in Src−/−;TRAMP mice and to a lesser extent, in Lyn−/−;TRAMP mice, was not due to the upregulation of other SFK members, but rather likely due to the loss of Src or Lyn. Fyn seemed not to be important for CaP initiation during the 16 to 34 weeks post-birth phase. In contrast, progression to NE disease, which is AR-independent, was not affected by the loss of Src, Lyn, or Fyn. This strengthens the notion that the role of SFK is to promote AR-driven AD disease, and indeed, AD-CaP initiation was delayed in TRAMP mice lacking Src or Lyn.
The loss of Src, and to a lesser extent the loss of Lyn, suppressed formation of NE micro- and macrometastases in various organs compared with TRAMP controls. Although in the case of Src−/−;TRAMP mice, this might be attributed to a severe delay in general disease onset, the dearth of metastases in the Lyn−/−;TRAMP group occurred at a period in which primary tumor formation was statistically comparable with that of TRAMP controls. This strongly suggests that Lyn, and possibly Src, play critical roles in metastasis formation in the TRAMP model.
An interesting phenomenon was noted, namely that a minority of late-stage Lyn−/−;TRAMP or Fyn−/−;TRAMP mice suffered from aggressive metastatic disease in the absence of primary-site tumors. These mice displayed prostatic hyperplasias or intraepithelial neoplasias, whereas their metastases were predominantly AD lesions based on staining for cytokeratins but not for synaptophysin. It is unclear whether Src also suppresses this process: only one Src−/−;TRAMP mouse, which was shown ultimately to lack a prostate tumor, presented with extensive AD metastases. This suggests that AD metastases arise from cells that disseminate early, and then, at a low frequency, progress oncogenically at distal sites. Moreover, the lack of primary-site tumors in these cases suggests some sort of suppressive cross-talk between the AD metastases and tumor-initiating cells in the prostate. Nonetheless, the mechanism by which Lyn and Fyn (and possibly, Src) inhibit formation of these AD metastases requires further analysis. Indeed, metastatic colonization (34), recruitment of endothelial cells to metastatic sites (35), and tumor invasiveness (36) require Src activity, whereas activation of Src pathways suppresses tumor dormancy, likely by favoring activation of ERK versus p38 MAPK pathways (37). This suggests that SFK might suppress the proliferation of already disseminated dormant cells, such that in SFK-null backgrounds, the incidence of AD metastases in mice lacking primary-site tumors increases. Thus, although the preponderance of our data suggests that the therapeutic targeting of SFK would prevent CaP initiation, progression, and metastasis formation based on the TRAMP model, there is worry that this might derepress the dormancy of disseminated AD-initiating tumor cells.
Disclosure of Potential Conflicts of Interest
I.H. Gelman is a consultant/advisory board member for Kinex Pharmaceuticals, LLC. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: I.H. Gelman, B.A. Foster
Development of methodology: I.H. Gelman
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): I.H. Gelman, J. Peresie, B.A. Foster
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): I.H. Gelman, K.H. Eng
Writing, review, and/or revision of the manuscript: I.H. Gelman, J. Peresie, K.H. Eng, B.A. Foster
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Peresie, B.A. Foster
Study supervision: I.H. Gelman, B.A. Foster
Grant Support
This study is supported by NIH and DoD funds, CA94108, PC061246, PC074228, PC101210, and W81XWH-11-2-0033 (to I.H. Gelman), and in part, by NIH/NCI Cancer Center Support Grant 2P30-CA016056 (including the Mouse Tumor Model, Laboratory Animal Research and Genomic Shared Resource Cores) and the National Functional Genomics Consortium.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Acknowledgments
The authors thank Sandra Buitrago Sexton, Aimee Stablewski, and Ellen Karasik for technical advice and assistance.
- Received September 12, 2013.
- Revision received June 16, 2014.
- Accepted July 4, 2014.
- ©2014 American Association for Cancer Research.
References
Volume 12, Issue 10
