The loss of TGF-beta signaling promotes prostate cancer metastasis

Abstract

In breast and colon cancers, transforming growth factor (TGF)-beta signaling initially has an antineoplastic effect, inhibiting tumor growth, but eventually exerts a proneoplastic effect, increasing motility and cancer spread. In prostate cancer, studies using human samples have correlated the loss of the TGF-beta type II receptor (T beta R II) with higher tumor grade. To determine the effect of an inhibited TGF-beta pathway on prostate cancer, we bred transgenic mice expressing the tumorigenic SV40 large T antigen in the prostate with transgenic mice expressing a dominant negative T beta R II mutant (DN II R) in the prostate. Transgene(s) and TGF-beta 1 expression were identified in the prostate and decreased protein levels of plasminogen activator inhibitor type I, as a marker for TGF-beta signaling, correlated with expression of the DN II R. Although the sizes of the neoplastic prostates were not enlarged, increased amounts of metastasis were observed in mice expressing both transgenes compared to age-matched control mice expressing only the large T antigen transgene. Our study demonstrates for the first time that a disruption of TGF-beta signaling in prostate cancer plays a causal role in promoting tumor metastasis.

Figure 1

Detection of transgene in transgenic mice. (A) An example of PCR genotyping using primer sets for the Tag transgene. The expected size of the Tag-amplified product is 430 bp. Transgene was detected in the 12T-7f/MT-DNIIR sample but not in the MT-DNIIR sample. (B) An example of PCR genotyping using primer sets for the DNIIR transgene. The expected size of the DNIIR-amplified product is 506 bp. Transgene was detected in the 12T-7f/MT-DNIIR sample but not in the 12T-7f sample.

Figure 2

Illustrations of histopathology of transgenic animals (H&E). (A) NHA in a DLP of a 12-week-old NT mouse. (B) LGPIN (arrow) in a DLP of a 23-week-old MT-DNIIR mouse. (C) HGPIN (arrow) in a DLP of an 18-week-old 12T-7 mouse. (D) MI (arrow) in a DLP of a 9-month-old 12T-10 mouse. (E) IC (arrow) in a DLP of a 20-week-old 12T-7f/MT-DNIIR mouse. (F) UC (arrow) in a VP of a 10-month-old 12T-10/MT-DNIIR mouse.

Figure 3

Detection of transgene expression in transgenic mice. (A) An example of immunohistochemical analysis shows expression of Tag in an HGPIN lesion (arrow) in a DLP of a 12T-7f/MT-DNIIR mouse. (B) A light field view of in situ hybridization shows expression of DNIIR in an HGPIN lesion (arrow) in a DLP of a 12T-7f/MT-DNIIR mouse. (C) An example of immunohistochemical analysis shows localization of TGF-β1 in an HGPIN lesion (arrow) in a DLP of a 12T-7f mouse.

Figure 4

Quantitation and normalization of PAI-I levels in transgenic mice. (A) Western blots for PAI-I and for β-actin as a loading control in 12T-7f and 12T-7f/MTDNIIR mice. (B) Summary of band intensities from Western blots in (A) along with ratios of PAI-I to β-actin. The average ratio is significantly lower in 12T-7f/MT-DNIIR mice than in 12T-7f mice (P < .05). (C) Western blots for PAI-I and for β-actin as a loading control in 12T-10 and 12T-10/MT-DNIIR mice. (B) Summary of band intensities from Western blots in (C) along with ratios of PAI-I to β-actin. The average ratio is significantly lower in 12T-10/MT-DNIIR mice than in 12T-10 mice (P < .05).

Figure 5

Metastatic lesions along with immunohistochemical analyses in 12T-7f/MT-DNIIR mice and 12T-7f mice. (A) Tag-expressing metastatic lesion (arrow) in the para-aortic lymph node of a 12T-7f/MT-DNIIR mouse. (B) TGF-β1-expressing metastatic lesion (arrow) in the lung of a 12T-7 mouse. (C) Metastatic lesion (arrow) on H&E stain in the liver of a 12T-7 mouse. (D) CG-expressing metastatic lesion (arrow) in the lumbar spine of a 12T-7f mouse. (E) Micrometastases (arrow) in the liver of a 20-week-old 12T-7f mouse. (F) Extensive micrometastases (arrow) in the liver of an 18-week-old 12T-7f/MT-DNIIR mouse.

Figure 6

Comparison of metastasis in 12T-10/MT-DNIIR mice and 12T-10 mice. (A and B) Gross metastatic lesions (arrow) in the liver of a 12T-10/MT-DNIIR mouse and accompanying histology. (C and D) No gross lesions in the liver of an age-matched 12T-10 mouse and accompanying histology. (E) Comparison of incidence (%) of gross metastasis over time in 12T-10/MT-DNIIR (●, n = 22) mice versus 12T-10 (■, n = 23) mice. The incidence ratio of metastasis is significantly greater in 12T-10/MT-DNIIR mice than in 12T-10 mice (P < .05).