Breast cancer is the leading cause of cancer death in women in the world, with most breast cancer morbidity and mortality resulting from metastatic disease (38
). Although the TGF-β signaling pathway has an important role in mammary carcinogenesis, the major components of the pathway, including the signaling receptors, TβRII and TβRI, and the predominant signaling pathway downstream of these receptors, Smad2, Smad3, and Smad4, are usually intact in human breast cancers (6
). In the present study, we demonstrate that expression of the TGF-β coreceptor TβRIII was frequently decreased at the mRNA and protein levels in human breast cancer, with approximately 90% of specimens demonstrating decrease or loss at the mRNA level and approximately 70% demonstrating decrease or loss at the protein level. Thus, we believe loss of TβRIII expression to be the most common alteration in the TGF-β signaling pathway described in human breast cancer to date. We have further demonstrated that loss of TβRIII expression was an early event, occurring initially in the preinvasive state, DCIS, with degree of loss correlating with breast cancer progression and corresponding to a decrease in patient survival. Mechanisms for decreased expression include LOH at the TGFBR3
gene locus and potential transcriptional downregulation of TβRIII by elevated TGF-β levels in the breast tumor microenvironment. Finally, we established a functional role for loss of TβRIII expression, as restoring TβRIII expression dramatically inhibited tumor invasiveness in vitro and tumor invasion, angiogenesis, and metastasis in vivo. Mechanistically, TβRIII appeared to function by undergoing ectodomain shedding, with sTβRIII antagonizing TGF-β signaling and reducing invasiveness and angiogenesis in vivo. Taken together, these results support loss of TβRIII expression as a frequent and important step in breast cancer progression, directly promoting breast cancer invasion and metastasis.
The dichotomous role of TGF-β signaling in breast cancer development has been experimentally verified in several murine models. Specifically, blocking TGF-β signaling in a series of human breast-derived cell lines representing different stages in breast cancer progression rendered premalignant cells tumorigenic, and low-grade tumorigenic cells more invasive, while making high-grade tumorigenic cells less metastatic (39
). In addition, introduction of constitutively active TβRI delayed oncogenic Neu-induced breast tumor onset but enhanced the frequency of lung metastasis in transgenic mice, whereas dominant-negative TβRII enhanced Neu-induced tumor onset but decreased subsequent lung metastasis (40
). Furthermore, inducing expression of active TGF-β1 after primary breast tumor formation dramatically enhanced lung metastasis in a murine breast cancer model without a detectable effect on primary tumor size (41
). Taken together, the results of these studies suggest that TGF-β suppresses breast cancer progression in the early stages, but enhances tumor progression and metastasis in the later stages. Different explanations for this dichotomous function have been proposed, including TGF-β exerting tumor-suppressing effects on epithelial-derived tumor cells and tumor-promoting effects on stromal cells (increased angiogenesis and immunosuppression, altered tumor cell–extracellular matrix interactions to enhance invasion and metastasis) (6
). However, emerging evidence suggests that TGF-β may exert its dichotomous effects during carcinogenesis at least in part through biphasic effects on the epithelial derived cancer cells themselves, as the cells alter their molecular profiles to differentially respond to TGF-β (6
). Thus, even though resistant to the tumor suppressor effects of TGF-β during tumorigenesis (growth inhibition, apoptosis, and differentiation), the cancer cells may respond to TGF-β with increased motility and invasiveness. Based on the present findings, we propose that loss of TβRIII expression may be a mechanism for this differential response to TGF-β during mammary carcinogenesis.
How might loss of TβRIII expression alter cellular responses to TGF-β during mammary carcinogenesis? Although TβRIII was the first TGF-β receptor cloned, as it has a short cytoplasmic domain with no intrinsic kinase activity, its role in TGF-β signaling has not been well characterized. TβRIII has classically been thought to act as a TGF-β coreceptor, concentrating ligand on the cell surface and enhancing ligand binding to the signaling TGF-β receptor TβRII (42
). However, emerging evidence supports a more substantial role for TβRIII in regulating and mediating TGF-β signaling. TβRIII has essential roles in chick (12
) and murine development, with the TβRIII knockout mouse having an embryonic lethal phenotype (13
). In addition, we have previously established that regulating TβRIII expression is sufficient to alter TGF-β signaling (26
), that the short cytoplasmic domain of TβRIII is phosphorylated by TβRII (14
) and interacts with the PDZ domain–containing protein GIPC to stabilize TβRIII expression on the cell surface and increasing TGF-β signaling (26
) as well as with the scaffolding protein β-arrestin2 to mediate internalization of TβRIII and TβRII and downregulation of TGF-β signaling (15
). In addition, TβRIII undergoes ectodomain shedding that releases the soluble extracellular domain (sTβRIII), which has been demonstrated to effectively neutralize TGF-β and antagonize autocrine TGF-β signaling. In breast cancer models, expressing sTβRIII has been demonstrated to decrease tumorigenicity and spontaneous lung metastasis in immunocompromised mice through effects on both the tumor cells (decreasing cell growth and increasing apoptosis) (43
) and the stroma (decreasing angiogenesis) (28
). While our results in the immunocompetent 4T1 model confirm the effects of TβRIII on angiogenesis, we found no significant effect of TβRIII expression on cellular proliferation or apoptosis in either primary tumor or distant tumor metastastic lesions in vivo. Instead, in addition to decreased angiogenesis, the major effect of TβRIII in vitro and in vivo was to decrease cellular invasiveness, with this effect mediated at least in part through the production of sTβRIII. Therefore, we propose a model in which loss of TβRIII expression results in alterations in TGF-β responsiveness in both a cell-autonomous fashion (resulting in relative resistance of breast cancer cells to TGF-β) and a non–cell-autonomous fashion (by decreasing production of sTβRIII), effectively increasing TGF-β signaling in both the cancer cells and the stromal elements. Our in vitro and in vivo results demonstrating decreased Smad2 phosphorylation and decreased TGF-β responsiveness in the presence of TβRIII suggest that non–cell-autonomous regulation by sTβRIII may have a dominant role in both tumor and stromal compartments. The contribution of TβRIII and sTβRIII on the balance of TGF-β signaling and responsiveness in epithelial and stromal compartments remains an area of active investigation.
TβRIII is located on chromosome 1p32, a region that frequently exhibits LOH in a wide variety of human cancers, including breast, colon, endometrial, gastric, kidney, lung, ovarian, and testicular cancer (20
). For breast cancer, LOH at 1p32 is associated with a poorer prognosis (20
). Previous studies have examined several potential tumor suppressor genes in this region, including mammary-derived growth inhibitor (44
) and TP73 (45
); however, expression and functional studies did not provide sufficient evidence supporting their role as tumor suppressor genes in breast cancer. In the present study, LOH analysis revealed allelic imbalance at the TβRIII loci in 50% of the patients, with LOH correlating with loss of TβRIII expression. The observed decrease in TβRIII mRNA and protein expression could result from haploid insufficiency, as previously reported for TGF-β1 (46
), or from transcriptional downregulation or promoter hypermethylation of the remaining allele. The current data strongly support TβRIII as a suppressor of breast cancer progression. TβRIII has also been reported to be lost at an early stage in renal cell carcinogenesis (47
). Whether TβRIII functions as a suppressor of cancer progression in renal cell and other human cancers remains to be discerned.
Although breast cancer is thought to progress from a preinvasive state (DCIS) to invasive disease, we currently cannot determine which DCIS lesions are likely to remain indolent, and thus may be treated by local resection only, versus those DCIS lesions that will progress to invasive disease and/or recur, necessitating more aggressive treatment (i.e., postresection radiation, mastectomy, or adjuvant hormonal or chemotherapy). Clearly, understanding the molecular mechanisms by which DCIS becomes invasive and ultimately metastatic will allow identification of patients at low or high risk of recurrence and invasion/metastasis and guide these treatment options. In the present study, our data support loss of TβRIII expression in DCIS as a common event potentially resulting in invasive and metastatic disease. Thus, as would be predicted, later-stage invasive cancers have a significantly higher frequency of TβRIII loss, and lower TβRIII expression correlates with a poorer prognosis for patients with invasive breast cancer. As this retrospective analysis was performed on patient tumor samples that were heterogeneous for both tumor and surrounding stromal tissue, we cannot be certain whether the loss in TβRIII expression was in the tumor, stroma, or both, although our own IHC analysis of a large tissue array support that loss was primarily in tumor cells. Whether TβRIII-negative DCIS lesions have a worse natural history and thus warrant more aggressive intervention than TβRIII-positive DCIS lesions requires prospective validation.