Perturbations are produced, but cells develop resistance to

Perturbations of the Transforming growth factor-? (TGF-?) signaling are central to tumorigenesis
through their effects on cellular process, including cell proliferation, extracellular matrix accumulation
and cell invasion. A large body of work established prominent roles for TGF-?s as important
mediators of epithelial to mesenchymal transition (EMT) which contributes to the degeneration of
mature epithelial structures and to the accumulation of extracellular matrix (ECM) during tumor
progression1. Thrombospondin 1 (TSP1) is a key regulator of latent TGF-? activation; emerging
evidence supports the concept of a major role for a TGF??–TSP1 axis in both in cancer and fibrotic
diseases2. In advanced cancers, a commonly observed but poorly understood mechanism is the loss of
tumor suppressive TGF-? signaling. Abnormally high levels of TGF-? are produced, but cells develop
resistance to TGF-? mediated growth arrest, allowing the pro-tumorigenic functions (e.g. EMT, stem-
like features and angiogenesis) of TGF-? to dominate3. We recently reported that suppression of latent
TGF-?1 by RNA interference in certain cancer cell types restores growth inhibitory TGF-? signaling
by disrupting a feedback loop involving microRNA-mediated regulation of the very factors (e.g.
TSP1) responsible for the activation of TGF-?1 from its latent precursor4-5. These studies identified
factors (siRNAs and miRNAs) which activate (TGF-?1 siRNAs) and repress (miR-18a) autocrine
growth inhibitory TGF-? signaling. Thus, a rescue of the TGF-? cytostatic response may be
achievable pharmacologically using oligonucleotides against TGF-?1 mRNA or miRNAs. This
implies that, paradoxically, TGF-?1 mRNA may represent a valid target for certain cancers, a central
theme in this proposal.
I propose to test a variety of oligoribonucleotides for their therapeutic properties. Globally, RNA-
based ligands will be designed, characterized and tested in cell culture systems and in vivo models.
Specifically, short interfering RNAs (siRNAs; a class of oligoribonucleotide drugs) will be used to
target latent TGF-?1 messenger RNA and associated factors. This will help to determine whether the
aforementioned mechanism of resistance identified in some cancer cell types also operates in in vivo
settings. This approach to targeting TGF-? is distinguished from other TGF-? targeting programs in
clinical development because it addresses the latent form of the cytokine in an effort to rescue the
tumor suppressive arm of the signaling pathway. Additional deliverables of the project include
information on other potential targets linked to the TGF-? biology, including a number of microRNAs
with potentially important roles in such diseases. A main deliverable of the project will be a proof-of
concept for the efficacy of this approach to TGF-? targeting in the preclinical setting. These studies
may lead to relevant advances in the current strategies to control expression and function of a critical
pathway implicated in various diseases through the development of effective RNA-based drugs.


We Will Write a Custom Essay Specifically
For You For Only $13.90/page!

order now

TGF-? polypeptides are cytokines which intricately control proliferation, differentiation, and survival
processes. Most cells synthesize TGF-? in excess and their rate-limiting mechanisms of activation are
regulated in stimulation-specific fashion6. TGF-?1 is expressed as a pro-peptide comprising the mature
form and a latency-associated peptide (LAP). The pro-peptide dimerizes and is nicked before secretion
by furin-like proteases but remains self-associated. Secretion is promoted after conjugation of a latent
TGF-?-binding protein (LTBP) to the LAP forming a large latent complex (LLC), which associates
with the extracellular matrix. Mature TGF-? is released from the LLC in vivo by cell surface proteases
and extracellular matrix proteins such as thrombospondin-1 (TSP1) and integrins that promote
dissociation of mature ligand. The mechanisms of activation, and also the type of intracellular
signaling, may depend on the TGF-?-containing complex bound to the cell surface. Consequently,
cells may respond differently to TGF-? from autocrine or exogenous origin by activating, for example,
alternative growth inhibitory pathways7. By maintaining a source of latent TGF-? close to its site of
action, cells can initiate rapid signaling without the need for new protein synthesis8. Once activated,
TGF-? binds a membrane-bound serine/threonine receptor complex (T?RI/T?RII), which

phosphorylates various substrates including Smad transcription factors, which accumulate in nuclear
complexes with co-activators and co-repressors, or molecules from numerous non-Smad pathways9.
TGF-? signaling via SMADs causes growth inhibition of epithelial cells by transcriptional induction
of cyclin-dependent kinase inhibitors P21 and P15, and the repression of transcription factors MYC,
ID1, and ID210. TGF-? signaling has been implicated in cancer since its discovery11. The loss of TGF-
? tumor suppressor function in human cancer from genetic mutations of components of the TGF?
signaling pathway results in uncontrolled cell proliferation. However, T?RII, T?RI and Smad2 are
mutated in several tumor types albeit with a low incidence (<5%)12 implying that other mechanisms lie behind these cancers. A commonly observed mechanism in cancer is the subversion of the tumor suppression arm of TGF-? signaling. Although abnormally high levels of TGF-? are produced, there is a loss of sensitivity (or resistance) to TGF-? mediated growth arrest3 whereas some responses to TGF- ? treatment e.g. SMAD2 phosphorylation and activation of pro-tumorigenic pathways, are maintained. The molecular mechanisms by which this resistance is achieved have not been fully characterized. One working hypothesis is that hyperactive TGF-? pathway might provide a selective advantage to the tumor that acquires high TGF-? expression. Indeed, high levels of TGF-? in the tumor and the circulation have been associated with poor prognosis13. Owing to the importance of TGF-? in control of tissue homeostasis, blockade of TGF-?1 synthesis might induce toxicity in some circumstances. The clinical investigators are cautious with use of anti- TGF-? drugs. Deletion of genes in the TGF-? pathway in mice disrupts vascular development14 and diseases caused by mutations in T?RI or T?RII such as the Marfan and Loeys-Dietz syndromes involve components of vascular abnormality and aneurysms15. Furthermore, a potential danger in using TGF-? antagonists in cancer patients is the potential to induce malignant transformation of premalignant cells. However, there is good reason to be optimistic: some pre-existing drugs are known to inhibit TGF-?, including pirfenidone, approved recently for idiopathic pulmonary fibrosis. In a three phase 3 trials, pirfenidone, an oral antifibrotic therapy, reduced disease progression. Treatment was associated with an acceptable side effect profile16. Similarly this proposed project will attempt to investigate RNA based pharmacological compounds that are capable of rescuing the normal TGF-? signaling.