SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression

Abstract

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Figure 1. SMAD4 is a putative suppressor of prostate tumour progression

a, b, Immunohistochemical (a) and western blot analysis (b) of wild-type (WT) and Pten^pc−/− use prostate tissues. Scale bar, 50 μm. c, Oncomine boxed plot of SMAD4 expression levels between human PCA and metastasis in multiple data sets including those from ref. 19 and ref. 20. d, SMAD4 knockdown enhanced metastatic potential to lung from PC3 cells implanted in renal capsule of immunocompromised nude mice.

Figure 2. Smad4 deletion drives progression of Pten-deficient prostate tumour to highly aggressive prostate cancer metastatic to lymph node and lung

a, Haematoxylin and eosin (H&E) stained sections of representative anterior prostates (AP) at 7, 11 and 15weeks. Scale bar, 200 μm. b, Kaplan– Meier cumulative survival analysis showing significant (P < 0.0001) decrease in

Figure 3. Ccnd1 and Spp1 are mediators of prostate tumour cell proliferation and metastasis

a, BrdU pulse-labelling and SA-β-galactosidase (β-Gal) staining of 15-week-old APs. b, Quantification of BrdU pulse labelling and β-Gal staining. Error bars represent s.d. for a representative experiment performed in triplicate. c, Western blot analysis demonstrating elevated Ccnd1 and Spp1 levels in Pten^pc−/− Smad4^pc−/− compared to Pten^pc−/− prostate tumours. d, Enforced CCND1 expression significantly enhanced prostate xenograft tumour growth of PC3 cells. e, f, Enforced SPP1 expression significantly increases metastatic activity of PC3 cells from prostate xenograft to lumbar lymph nodes (e) and to lung (f).

Figure 4. Prognostic potential of a four-gene signature in human PCA

a, The four-gene set of PTEN/SMAD4/CCND1/SPP1 can dichotomize PCA cases for BCR in the ref. 13 data set. b, c, C-statistic analysis revealed that this four-gene set can enhance the prognostic accuracy of Gleason score in the ref. 13 data set (b) and in an independent PHS cohort (c). d, TMA-based four-protein model also significantly improve the prognostic ability of Gleason (P = 0.015) from the PHS cohort. e, Representative immunohistochemical staining with specific antibody against PTEN, SMAD4, CCND1 and SPP1 in the Directors Challenge TMA. Scale bar, 200 μm.