Browsing by Subject "TGF"

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 specification of a single axon and multiple dendrites is the first observable event during neuronal morphogenesis and such structural specialization underlies neural connectivity and nervous system function. Numerous intracellular signaling components that are required for axon specification have been described but how such signaling paradigms are initiated by extracellular factor(s) within the embryonic milieu is poorly understood. Here, I describe how transforming growth factor-β (TGF-β), an embryonic morphogen that directs structural plasticity and growth in various cell types, initiates signaling pathways both in vivo and in vitro to fate naïve neurites into axons. Using conditional knockout strategies, I found that cortical neurons lacking the type II TGF-β receptor (TβR2) fail to initiate axons during development, and interestingly, fail to engage radial migration. In cultured neurons, exogenous TGF-β is sufficient to direct the rapid growth and differentiation of an axon and genetic enhancement of receptor activity promotes the formation of multiple axons. The cellular polarization of receptor activity occurs through the interaction of the type-I TGF-β receptor with Par6, a component of the axon-specifying Par3/Par6 polarity complex. Receptor distribution is restricted to axons, and downstream signaling events required for axon specification are triggered when Par6 is phosphorylated by TβR2. Together, these results indicate that TGF-β is the extrinsic cue for neuronal polarity in vivo and directs neuronal polarity by controlling Par6 activity and cellular migration during axon generation.

Breast cancer remains among the most common cancers of the developed world. Despite advances in treatment modalities, deaths due to breast cancer are the second leading cause of cancer death among women. The transforming growth factor-beta (TGF-β) pathway is an important modulator of breast cancer progression, acting in a tumor suppressing fashion in early carcinogenesis but switching in a poorly understood fashion to a promoter of cancer progression in later stages. Mutations and loss of function of TGF-β components are common across a variety of cancers. In particular, the expression of the type III TGF-β receptor (TβRIII) is decreased with cancer grade and clinical progression in prostate, lung, ovarian, and pancreatic cancers. In an effort to enhance our understanding of the biology of TGF-β on carcinogenesis, this dissertation looks at the role of TβRIII in breast cancer progression.

Through an examination of clinical specimens, loss of TβRIII was seen at both the message and protein levels with increasing tumor grade. Analysis of correlated patient outcomes showed that low TβRIII expression was predictive of a shorter time to recurrence, demonstrating clinical relevance for TβRIII expression. The contribution of TβRIII to tumor progression was further examined by examining known TGF-β functions, including proliferation, apoptosis, migration, and invasion. TβRIII had no effect on proliferation or apoptosis, but had a suppressive effect on metastasis in vivo, as mammary cancer cells stably expressing TβRIII that were orthotopically injected exhibited lower metatstatic burden and local invasion. In vitro, breast cancer cells exhibited suppression of migration and invasion in transwell assays. Finally, soluble TβRIII (sTβRIII) was shown to recapitulate the suppressive effects on invasion.

To further explore other potential mechanisms by which TβRIII may be mediating its tumor suppressive effects, I examined the contribution of the cytoplasmic domain of TβRIII, which is known to be critical in the regulation of TβRIII cell surface expression and downstream signaling. In vitro, I demonstrated that abrogation of the cytoplasmic domain attenuates the TβRIII-mediated suppression of migration and invasion. TβRIII's suppressive effects are also concomitant with loss of TGF-β signaling, as abrogation of the cytoplasmic domain failed to attenuate TGF-β signaling while the full length receptor was able to do so. In vivo, I also showed that in the absence of the cytoplasmic domain, TβRIII is unable to suppress metastasis and local invasion. Finally, a closer dissection of the cytoplasmic domain revealed that abolishing the interaction of TβRIII with the scaffolding protein GIPC also attenuated TβRIII's ability to dampen TGF-β signaling and invasion.

In sum, TβRIII was established as a prognostic marker for recurrence-free survival of breast cancer patients and as a suppressor of metastasis, migration, and invasion. Furthermore, several mechanisms contribute to TβRIII's tumor suppressive effects, namely the generation of sTβRIII and the interaction of TβRIII with GIPC. Taken together, these studies further demonstrate the importance of TGF-β signaling in cancer biology, elucidate mechanisms by which TβRIII suppresses breast carcinogenesis, and expand upon our understanding of the emerging roles of TβRIII in regulating tumor biology in general.