Browsing by Subject "Smad"

Transforming growth factor–β (TGF–β) signaling regulates a range of processes in a variety of cell types. Consequently, TGF–β plays a complex role in the progression of several types of cancers; it acts as a tumor suppressor in normal cells and early in tumor progression, yet it can promote tumor progression in later stages of cancer.

Among the cancers that TGF–β has been implicated in is glioblastoma multiforme (GBM), the most common primary brain neoplasm and one of the most lethal types of cancer. Because of its high mortality rate and the lack of effective treatments, discovering the molecular mechanisms that underlie GBM formation and growth is of great clinical interest. To this end, we investigated the function of a TGF–β target gene — the putative tumor suppressor N‐Myc downstream‐regulated gene 4 (NDRG4) — in GBM cell viability, proliferation and tumor formation. Contrary to the established roles of other NDRG family members, we found that NDRG4 expression is elevated in GBM and that NDRG4 is required for the survival of established GBM cell lines and primary GBM xenograft cells enriched for highly tumorigenic GBM cancer stem cells. Knockdown of NDRG4 expression results in G₁ cell cycle arrest followed by apoptosis that is associated with a decrease in the expression of XIAP and survivin. Finally, knockdown of NDRG4 expression in established GBM cell lines and GBM cancer stem cells results in decreased tumorigenicity following intracranial implantation of these cells into immunocompromised mice. Collectively, these data indicate that NDRG4 does not function as a tumor suppressor like other NDRG family members, but rather it is essential for GBM tumorigenicity and may represent a potential therapeutic target for this devastating disease.

In the second portion of this dissertation, we examine the TGF–β cytostatic signaling pathway in B lymphocytes. TGF–β–induced growth inhibition is the most extensively studied biological response to a TGF–β signal. Although in most cell types this response is mediated by Smad3– dependent regulation of c–Myc, p15Ink4B, and p21Cip1 transcription, studies from Smad3 null mice suggest that TGF–β–induced growth inhibition in B lymphocytes occurs regardless of Smad3 status. We prove that this response does indeed occur independently of Smad3 in purified primary B lymphocytes and WEHI–231 cells. Consistent with this, p15Ink4B and p21Cip1 are not noticeably induced by TGF–β in these cells, whereas Id3 and cyclin G2 are induced in a Smad3–independent manner. Finally, unlike the MAPK pathways we tested, the BMP–specific Smads 1 and 5 are activated in response to TGF–β in these cells, and this activation is dependent on ALK5 kinase activity. Collectively, these data indicate that TGF–β induces growth inhibition in B lymphocytes through a novel signaling pathway, and Smads 1 and 5 may help mediate this response.

The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and homeostasis. BMPs have been shown to pattern the dorsoventral axis in zebrafish (Danio rerio) early during embryonic development by establishing a dorsal-to-­ventral ligand gradient. During tumorigenesis, BMPs primarily function as tumor promoters, as an increase in BMP expression is associated with an increase in invasion, migration, epithelial-­to-­mesenchymal transition (EMT), proliferation and angiogenesis.

Although it is clear that BMPs play multiple roles in these biological events, the precise mechanism by which BMPs mediate these functions is not fully understood. Canonically, BMP ligands signal through cell surface receptor complexes that phosphorylate transcription factors, Smad1, Smad5 and Smad8, which mediate BMP-­ specific gene transcription. While studying BMP signaling during cancer progression, we determined that BMPs unexpectedly signal through the canonical TGF-β-­responsive transcription factors, Smad2 and Smad3.

We determined that BMP-­induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP binding TGF-­β superfamily receptors, ALK3/6, and the Smad2/3 phosphorylating receptors, ALK5/7. BMP signaling through Smad1/5/8 induces MET, while Smad1/5 and Smad2 mediate dorsoventral axis patterning in zebrafish embryos and Smad3 facilitates invasion.

Taken together, our data provides evidence that BMP-­induced Smad2 and Smad3 phosphorylation occurs through a non-­canonical signaling mechanism to mediate multiple biological events. Thus, the signaling mechanisms utilized by BMPs and TGF-­β superfamily receptors are broader than previously appreciated.