The development and maintenance of multicellular organisms requires tight control over the proliferation, differentiation, movement, organization, and death of their constituent cells. Intricate molecular communication networks have evolved to control these processes. Our work is focusing on how cells receive, read, and relay such signals, and how disruptions in these processes lead to tumor formation and cancer metastasis. We are approaching these questions through the rich venue provided by the transforming growth factor-β (TGFβ) pathway, as well as through the direct identification of metastasis genes and functions.
With nearly 40 related members in the human genome, TGFβ represents one of the most prominent and functionally versatile families of cytokines. This family includes the TGFβs, nodals, activins, bone morphogenetic proteins (BMPs), myostatins, anti-Müllerian hormone, and other factors. These factors have profound effects on essentially every aspect of cell behavior. Produced by many cell types or restricted to just a few, they guide early stages of embryo development and tissue homeostasis throughout life.
We uncovered key steps of this signaling pathway by identifying the TGFβ and BMP receptors and elucidating their mechanism of activation. The receptors are serine/threonine protein kinases, and their substrates are the Smad transcription factors. We showed that Smad C-terminal phosphorylation by TGFβ and BMP receptor kinases, and Smad linker phosphorylation by MAPK, GSK3, and other protein kinases, are key events in Smad regulation. We recently found that Smad C-terminal phosphorylation creates a binding site for cofactors, including Smad4 and TIF1γ, assembling transcriptional complexes that form different branches in the TGFβ pathway. Moreover, linker phosphorylation couples the Smad pathway to regulatory inputs by creating docking sites for Smad ubiquitin ligases of the Smurf family. Smad linker phosphorylation is reversed by SCP1–3 phosphatases, which also play a role in Smad C-terminal dephosphorylation. These findings are paving the way for new discoveries on the function and network integration of the TGFβ and BMP pathways.
The cellular response to TGFβ depends on the cell type and the context. We propose that activated Smad factors regulate different genes in different ways in different cell types by associating with diverse DNA-binding cofactors, whose availability depends on the cell type. These interactions generate distinct repertoires of Smad transcriptional complexes, each complex targeting a small set of genes for either activation or repression. With this in mind, we recently identified FoxO, C/EBPβ, E2F4/5, and ATF3 as the Smad cofactors that mediate, respectively, the induction of p15INK4 and p21CIP1 and the repression of c-MYC and ID1. These gene responses underlie one of the most important effects of TGFβ, namely, growth inhibition.
TGFβ is the most prominent cytokine inhibitor of cell proliferation. This effect involves the repression of cell proliferation genes, such as c-MYC and ID1, and the induction of cyclin-dependent kinase inhibitors, two of which—p27Kip1 and p57Kip2—we codiscovered. Besides providing a coherent program for cell cycle inhibition, these transcriptional mechanisms are allowing us to identify alterations that are responsible for the evasion of the TGFβ cytostatic action in breast cancer and brain tumors. Moreover, once relieved from the growth inhibitory action, tumor cells may use TGFβ to escape immune surveillance. We recently found that tumor-derived TGFβ inhibits the expression of five tumor cell-killing genes in cytotoxic T lymphocytes. And, not content with losing TGFβ growth inhibitory responses, tumor cells may alter the Smad pathway to undertake metastasis to the bones and lungs in breast cancer patients. We are identifying the TGFβ target genes and mechanisms that mediate metastasis to these organs. In sum, as tumors develop they turn TGFβ from a tumor-suppressor signal into an accomplice in metastasis.
Metastasis is the cause of 90 percent of deaths from cancer, and yet little is known about its underlying mechanisms. We are addressing this problem by exploiting the fact that tumors have distinct patterns of organ-specific colonization, each with a distinct biology and clinical evolution. We are identifying clinically relevant genes that mediate tumor microenvironment interactions, cancer cell entry and exit from the circulation, and cancer stem cell colonization of various organs. By combining in vivo selection of human metastatic cells, transcriptomic profiling, and functional testing, we have identified genes that selectively mediate breast cancer metastasis to bones or lungs. Gene transfer and gene-silencing techniques indicate that some of these genes serve dual functions, providing growth advantages both in the primary tumor and in the metastasis microenvironment. Others contribute to aggressive growth selectively in a particular organ. Bioinformatics analysis of hundreds of primary tumors has resulted in a lung metastasis gene expression signature (LMS) that predicts clinical outcome in patients with estrogen receptor–negative breast cancer. We are investigating the role of the LMS in other tumors that metastasize to the lungs.
Using genetic and pharmacological approaches, we recently found that several LMS genes, including the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases MMP1 and MMP2, collectively facilitate the assembly of new tumor blood vessels, the release of tumor cells into the circulation, and the breaching of lung capillaries by circulating tumor cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.
