TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression

Abstract

The TGF-beta signaling network plays a complex role in carcinogenesis because it has the potential to act as either a tumor suppressor or a pro-oncogenic pathway. Currently, it is not known whether TGF-beta can switch from tumor suppressor to pro-oncogenic factor during the course of carcinogenic progression in a single cell lineage with a defined initiating oncogenic event or whether the specific nature of the response is determined by cell type and molecular etiology. To address this question, we have introduced a dominant negative type II TGF-beta receptor into a series of genetically related human breast-derived cell lines representing different stages in the progression process. We show that decreased TGF-beta responsiveness alone cannot initiate tumorigenesis but that it can cooperate with an initiating oncogenic lesion to make a premalignant breast cell tumorigenic and a low-grade tumorigenic cell line histologically and proliferatively more aggressive. In a high-grade tumorigenic cell line, however, reduced TGF-beta responsiveness has no effect on primary tumorigenesis but significantly decreases metastasis. Our results demonstrate a causal role for loss of TGF-beta responsiveness in promoting breast cancer progression up to the stage of advanced, histologically aggressive, but nonmetastatic disease and suggest that at that point TGF-beta switches from tumor suppressor to prometastatic factor.

Figures

Figure 1

TGF-β receptor expression and responsiveness in the four human breast epithelial cell lines. (a) Ligand affinity cross-linking. Cell lines were affinity labeled with 100 pM 125I-labeled TGF-β1 in the absence (–) or presence (+) of a 50-fold molar excess of unlabeled TGF-β1. The migration positions of the endogenous TGF-β receptors (RI, RII, and RIII) are indicated. MW, molecular weight. (b) Smad phosphorylation and regulation of gene expression. Cells were treated with 2 ng/ml TGF-β and assessed by Western blot analysis for extent of Smad phosphorylation (at t =15 minutes), and effects on fibronectin (FBN) and c-Myc expression (at t = 24 hours). For the c-Myc blot, the arrow indicates the c-Myc band and the filled square indicates a nonspecific band. (c) Growth-inhibitory effects of TGF-β1. Cell proliferation in the presence of increasing amounts of TGF-β1 was quantitated by 3H-thymidine incorporation (incorp.). Results are the means for three determinations normalized to the controls with no added TGF-β for each cell type.

Figure 2

Blockade of TGF-β responses in vitro by transduction with the DNR. (a) Expression of the DNR in transduced cells. DNR expression in transduced M-III cells was determined by Western blot analysis probing for the Myc tag on the DNR. β-Actin protein was used for normalization. (b) Ability of the DNR to bind TGF-β. M-III cells were affinity labeled with 125I-TGF-β1. Following cross-linking, lysates were immunoprecipitated with anti-Myc Ab for visualization of ligand bound to the DNR. (c) Effect of DNR on Smad phosphorylation by TGF-β. M-III cells were treated with 5 ng/ml TGF-β1 for various times, and Smad protein expression and phosphorylation were analyzed by Western blot. P-Smad, phosphos-Smad. (d) Effect of DNR on gene-regulation responses to TGF-β1. M-III cells were treated with 5 ng/ml TGF-β1 or vehicle alone for 18 hours, and fibronectin (FBN) and c-Myc expression were analyzed by Western blot. (e) Effect of DNR on growth inhibition induced by TGF-β1. Growth inhibition in response to TGF-β1 was measured by [3H]-thymidine incorporation. All results are the mean ± SD for three determinations and are normalized to no TGF-β controls for each sample. PAR, untransduced parental M-III cells; CON, M-III cells transduced with pLPCX control retrovirus; DNR, M-III cells transduced with pLPC-DNR.

Figure 3

Decreased TGF-β responsiveness increases the probability of malignant conversion for the premalignant breast cell line M-II. (a) TGF-β responsiveness of M-II transductants in vitro. The proliferation of M-II transductants in the presence (+) and absence (–) of 2 ng/ml TGF-β1 was determined by incorporation of 3H-thymidine. Results are the mean ± SD of three determinations and are normalized to the no TGF-β control in each case. *P < 0.01. (b) Tumor growth kinetics. Five-week-old female athymic nude mice were inoculated subcutaneously on each hind flank with retrovirally transduced M-II cells (5 × 106 cells/site; ten sites per genotype). (c) Histology of lesions formed by M-II transductants. M-II CON cells formed cystic ductal structures, while MII-DNR formed glandular carcinomas. CON, M-II cells transduced with pLPCX; DNR-pool, pooled M-II cells transduced with pLPC-DNR at levels that fully blocked TGF-β growth-inhibitory responses; DNR-c103, a clone of M-II cells transduced with pLPC-DNR that retained partial TGF-β responsiveness.

Figure 4

Decreased TGF-β responsiveness increases the tumor growth rate and histological grade of the low-grade breast carcinoma line M-III. (a) Tumor growth kinetics. Nude mice were inoculated subcutaneously on each hind flank with retrovirally transduced M-III cells (106 cells/site; 10 sites/genotype). Untransduced parental M-III cells (n = 4, not shown) gave results essentially identical to M-III CON. (b–e). Histology of lesions formed by M-III transductants. Tumors of both genotypes were admixtures of three morphologic types. (b) M-III DNR tumor showing all three morphologies (Cr, cribriform structures; Cl, clear cells; At, area of atypia); (c) cribriform glands in an M-III CON tumor; (d) clear cell area in an M-III CON tumor; (e) area of atypia from an M-III DNR tumor. Magnification: ×100 (b) and ×400 (c–e). (f) Atypia and mitosis grades in M-III tumors. Histological sections were graded from 0 to 4 independently for extent of atypia and for frequency of mitoses as detailed in Methods. (g) Proliferation and apoptosis rates in M-III tumors. Tumor cell proliferation was quantitated by counting BrdU-labeled nuclei on histologic sections, and apoptotic cells were quantitated by TUNEL assay. Results are the mean ± SD for a minimum of five tumors of each genotype. PAR, parental untransduced cells; CON, cells transduced with pLPCX; DNR, cells transduced with pLPC-DNR. hpf, high-power field.

Figure 5

Decreased TGF-β responsiveness does not affect primary tumorigenesis but suppresses metastasis in the high-grade breast carcinoma line M-IV. (a) Tumor growth kinetics in vivo. Nude mice were inoculated subcutaneously on each hind flank with retrovirally transduced M-IV cells (2 × 105 cells/site). For M-IV CON, n = 11 sites injected; M-IV DNR, n = 11. Untransduced parental M-IV cells gave essential identical results to M-IV CON (n = 4, not shown). (b) Lung metastases. Metastatic efficiency was determined by quantitation of histologically detectable lung metastases 8 weeks after injection of 106 retrovirally transduced cells into the tail vein of nude mice. Results are the mean ± SD for n = 5 (M-IV CON) and n = 10 (M-IV DNR). CON, cells transduced with pLPCX; DNR, cells transduced with pLPC-DNR.

Figure 6

Summary of effect of decreased TGF-β response at different stages in the carcinogenic process for the breast.