Abstract
BackgroundTGFβ signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFβ signaling.MethodsTo test the importance of TGFβ signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFβ response (FET), or tumorigenic with TGFβ response (FETα) or tumorigenic with abrogated TGFβ response via introduction of dominant negative TGFβRII (FETα/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging.ResultsAbrogation of TGFβ signaling through introduction of a dominant negative TGFβ receptor II (TGFβRII) in non-metastatic FETα human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFβ signaling in FETα-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFβ signaling is a metastasis suppressor, we rescued TGFβ signaling in CBS metastatic colon cancer cells that had lost TGFβ receptor expression due to epigenetic repression. Restoration of TGFβ signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFβ signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma.ConclusionsThe observations presented here indicate a metastasis suppressor role for TGFβ signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFβ signaling may be imprudent in some patient populations with residual TGFβ tumor suppressor activity.
Highlights
transforming growth factor-β (TGFβ) signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy
Abrogation of TGFβ signaling was confirmed by treating FETα and FETα-dominant negative (DN) cells with varying concentrations of TGF β [0, 5, 10 ng/mL] for 2 h followed by immunoblot analysis
To assess the effect of TGFβ treatment on cell survival, cells were treated in the presence or absence of 5 ng/mL TGFβ in combination with growth factor deprivation stress (GFDS) for 48 h followed by DNA fragmentation assays which showed a 3-fold increase in DNA fragmentation of FETα cells treated with TGFβ (Figure 3F). These results indicate that TGFβ mediated inhibition of survivin and X-linked inhibitor of apoptosis (XIAP) expression is associated with FETα cell sensitivity to apoptosis
Summary
TGFβ signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. Many cellular pathways have been linked to enhanced survival or anti-apoptotic signaling and malignant progression; here we investigated the role of transforming growth factor-β (TGFβ) in an orthotopic colorectal cancer model of metastasis. TGFβ signaling through Smad activation is regarded as tumor suppressive during the early stages of cancer and precancerous lesions as it has been shown that loss of TGFβ tumor suppressor signaling has been associated with tumor initiation and progression of several types of tumors including colon cancer. Other types of cancer as well as some subsets of colon cancer are often associated with epigenetic transcriptional repression of TGFβ receptors rather than mutational inactivation of the pathway [7,8,9], contributing to a loss in growth control as well as resistance to apoptosis [10,11]. One view is that TGFβ tumor promotion may occur predominantly in situations where signaling receptor expression is deficient [15]
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