FGFR1 is essential for prostate cancer progression and metastasis

Abstract

The fibroblast growth factor receptor 1 (FGFR1) is ectopically expressed in prostate carcinoma cells, but its functional contributions are undefined. In this study, we report the evaluation of a tissue-specific conditional deletion mutant generated in an ARR2PBi(Pbsn)-Cre/TRAMP/fgfr1(loxP/loxP) transgenic mouse model of prostate cancer. Mice lacking fgfr1, in prostate cells developed smaller tumors that also included distinct cancer foci still expressing fgfr1 indicating focal escape from gene excision. Tumors with confirmed fgfr1 deletion exhibited increased foci of early, well-differentiated cancer and phyllodes-type tumors, and tumors that escaped fgfr1 deletion primarily exhibited a poorly differentiated phenotype. Consistent with these phenotypes, mice carrying the fgfr1 null allele survived significantly longer than those without fgfr1 deletion. Most interestingly, all metastases were primarily negative for the fgfr1 null allele, exhibited high FGFR1 expression, and a neuroendocrine phenotype regardless of fgfr1 status in the primary tumors. Together, these results suggest a critical and permissive role of ectopic FGFR1 signaling in prostate tumorigenesis and particularly in mechanisms of metastasis.

Figure 1

In situ hybridization for localization of FGFR1 message in mouse prostate gland. FGFR1 message is localized exclusively in stroma in day 3 postnatal prostate gland (Panel A) and in normal prostate gland (shown as 12 week old, 200×, Panel B). Panel C: Elevated FGFR1 message in prostate epithelium in high grade PIN in the TRAMP prostate cancer mouse model (200×). Panel D: Elevated FGFR1 message in carcinoma cells in TRAMP tumors (200×). Panel E: Elevated FGFR1 message in carcinoma cells in metastatic TRAMP tumors (red arrow) as opposed to adjacent tissue (black arrow) (200×).

Figure 2

Conditional ablation of fgfr1 results in attenuated tumorigenesis and altered phenotype in the TRAMP mouse model. Panel A: an example of four mice where the prostate tissues / tumors from two are positive (KO+) and two are negative (KO−) for the fgfr1^KO allele. Panel B and C show an example histopathology (H&E section) and the corresponding in situ hybridization for FGFR1 message in a serial section respectively at foci PIN and well differentiated cancer exhibiting some focal escape from Cre-mediated excision (arrow) in a 22-week KO+ mouse (200×). Panels D and E show an example of escape of excision in foci of poorly differentiated cancer that are positive for FGFR1 message in the same mouse (200×). Panels F and G show universally positive FGFR1 message in poorly differentiated cancer in a 22-week KO− mouse (200×). Panel H: Knockout of fgfr1 results in a significant reduction in tumor mass in mice with detected fgfr1 KO allele (KO+) as compared to mice with undetected KO allele (KO−) or wild type controls. Numbers of mice (n) in each cohort are shown above the bars. Asterisks indicate significant changes (p<0.05). No TRAMP: mice that do not carry TRAMP transgene. Panel I shows the percent of mice in each cohort that contain the indicated histopathology phenotype (open bars are wild type controls).

Figure 3

Immunohistochemistry for Ki67. Panel A: Regions of well differentiated carcinoma in TRAMP control mice show a focal and diffuse pattern of Ki67 immunoreactivity. Panel B: Regions of poor differentiation in control TRAMP tumors exhibit a dense pattern of Ki67 immunoreactivity. Panel C: Mice with detected fgfr1 KO allele (KO+) exhibit an intermittent and diffuse pattern similar to control TRAMP mice with no regions of dense staining. Panel D: Mice with no detectable fgfr1 KO allele (KO−) exhibited poorly differentiated tumors that exhibited a dense Ki67 staining pattern, similar to the poorly differentiated tumors in the TRAMP control mice. Panels E and F. Primary tumor with a KO+ genotype (Panel E) showing intermittent and focal staining in comparison to a liver metastasis (Panel F) in this same mouse with dense and near universal staining of carcinoma cells. (100× all panels).

Figure 4

Ablation of fgfr1 results in altered mean time to death in TRAMP mice. Panel A: Mice with fgfr1 KO+ tumors (n=11) showed a significantly increased mean time-to-death (38 weeks) as compared to mice with fgfr1 KO− tumors (26.3 weeks) (n=11) (p=0.0172, Gehan-Breslow-Wilcoxon test). Panels B and C: HE staining and in situ hybridization for FGFR1 message show that fgfr1 KO+ tumors (shown as a phyllode tumor) exhibited foci with either no reactivity or some isolated foci with positive reactivity for FGFR1 (arrow) (200×). Tumors with a phyllodes histopathology were differentially observed in 75% of the fgfr1 KO+ tumors. Panels D and E: fgfr1 KO− tumors were poorly differentiated and universally positive for fgfr1 (200×). Only one of the KO− tumors carried minimal phyllodes-like foci and most were poorly differentiated. (200×). Panel F: Respective histopathology of tumors at time of death. Poorly differentiated tumor foci were observed in 100% of fgfr1 KO− tumors, whereas only 38% of fgfr1 KO+ tumors exhibited poorly differentiated foci.

Figure 5

Metastatic tumors do not exhibit the fgfr1 KO allele. Panel A: PCR of genomic DNA from metastatic tumors in 15 out of 16 ARR₂PBi-Cre/TRAMP/fgfr1^loxP/loxP mice examined in the survival study showed no fgfr1 KO allele in any of the metastatic tumor sites. Mouse No. 1201 is a representative mouse exhibiting no KO allele in primary tumor (fgfr1 KO−). Mice No. 1204, 1237 and 2288 are representative samples of mice with the KO allele in the primary tumor (fgfr1 KO+). Regardless, no KO allele was detected in metastases to lymph node, liver or lung. PCR for ARR2PBi-Cre transgene was used as control. Panel B and C shows representative in situ hybridization for FGFR1 message in primary tumors in fgfr1 KO+ mouse No. 2288 (200×). Note some focal escape from Cre excision shown in Panel C. Panels D and E shows the H&E section (D) and the corresponding in situ hybridization (E) from an adjacent serial section from a metastatic tumor to liver from the same mouse (200×). Panels F and G shows a similar association with metastatic tumor to the lung in the same mouse (200×). PT: primary prostate tumor, LN: lymph node metastasis, LUM: lung metastasis, LIM: liver metastasis, SV: seminal vesicle.