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A. Gulino - Regulation of the Hedgehog signaling in neural cell development and disease
Identification of novel Hedgehog-target genes
in cerebellar neural progenitors and
medulloblastoma
Cerebellar neural progenitor development occurs during
the first two weeks of post-natal life, under the
activity of Shh. Therefore, we first analyzed gene
expression profles in mouse cerebella during the first
two weeks of development by means of Gene-Chip
Murine microarray (Affimetrix), to identify subsets of
genes specifically upregulated in the time window in
which Hedgehog pathway is strongly activated. Then,
we have selected several of these genes and verified if
they are induced in cultured cerebellar GCPs treated
with Shh. Finally, we analyzed mouse and human medulloblastoma
samples for the expression levels of these
genes. Through this approach, we have identified Insm1
and Nhlh1/NSCL1 as new Hedgehog target genes, that
are induced by Shh in cultured GCPs. We have identified
and isolated Nhlh1 promoter and found that it is bound
and activated by Gli1 transcription factor. Remarkably,
the expression of these genes is also upregulated in
mouse and human Hedgehog–dependent medulloblastomas,
suggesting that they may be either a part of the
Hedgehog-induced tumorigenic process or a specific
trait of Hedgehog-dependent tumor cells (De Smaele et
al., Neoplasia 2008, 10:89-98).
Dissecting transcriptional function of AP-1
The AP-1 complex mediates the transcriptional
response to mitogens and its deregulation causes developmental
defects and tumors by cross-talking with a
number of signaling pathways including Hedgehog.
The molecular mechanisms of this cross-regulation are
poorly understood. We have observed that the coactivator
TORC1 (Transducer Of Regulated CREB 1, also
called CRTC1) is a potent and indispensable modulator
of AP-1 function. Following exposure of cells to the
AP-1 agonist TPA, TORC1 is recruited to AP-1 target
gene promoters and associates with c-Jun and c-Fos to
activate transcription. Consistently, TORC1 synergizes
with the proto-oncogene c-Jun to promote cellular
growth, whereas AP-1-dependent proliferation is abrogated
in TORC1-deficient cells. Remarkably, we demonstrate
that TORC1-Maml2 fusion oncoprotein binds
and activate both c-Jun and c-Fos. As a consequence,
ablation of AP-1 function disrupts cellular transformation
and proliferation mediated by this oncogene. Taken
together, these data illustrate a novel mechanism
required to couple mitogenic signals to the AP-1 gene
regulatory program (Canettieri et al., PNAS 2009).
A novel pro-apoptotic transcriptional function
of the retinoblastoma tumor suppressor protein
The function of the retinoblastoma tumor suppressor
protein (pRB) is disrupted in many human tumors
through either inactivation of the Rb gene or alter-
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ations in its upstream regulators. pRB’s loss of function
has been reported to cooperate with Hedgehog pathway
for cerebellar tumorigenesis via unelucidated mechanisms.
pRB’s tumor suppressive activity is at least partially
dependent upon its ability to arrest cells through
E2F transcription factors inhibition. This inhibition is
relieved through mitogen-induced phosphorylation of
pRB, triggering E2F release and activation of cell cycle
genes. We have previously reported that E2F1 can also
activate pro-apoptotic genes in response to genotoxic or
oncogenic stress, via acetylation-dependent protein
modification (Pediconi et al., Nat Cell Biol 2003, 5:552;
Ianari et al., J Biol Chem. 2004, 279:30830). However,
pRB's role in this context has not been established. We
have observed that DNA damage and E1A-induced
oncogenic stress promotes formation of a pRB-E2F1
complex even in proliferating cells. Moreover, pRB is
bound to pro-apoptotic promoters that are transcriptional
active and pRB is required for maximal apoptotic
response in vitro and in vivo. Together, these data
reveal a previously unappreciated function for pRB in
the induction of apoptosis in response to genotoxic or
oncogenic stress. Our data now establish a second role
for pRB as a stress-induced activator of apoptosis.
Notably, pRB’s ability to promote either arrest versus
apoptosis seems to be context dependent, with apoptosis
being favored in proliferating cells. This finding has
the potential to explain why cells are typically more
resistant to apoptosis when in the arrested state. Most
importantly, our observations suggest that Rb status
will influence tumor response to chemotherapy by
impairing both the arrest and apoptotic checkpoint
responses (Ianari et al., Cancer Cell 2009).
Selected publications
De Smaele E, Fragomeli C, Ferretti E, Pelloni M, Po
A, Canettieri G, Coni S, Di Marcotullio L, Greco A,
Moretti M, Di Rocco C, Pazzaglia S, Maroder M,
Screpanti I, Giannini G, Gulino A. An integrated
approach identifies Nhlh1 and Insm1 as Sonic
Hedgehog-regulated genes in developing cerebellum
and medulloblastoma. Neoplasia 2008, 10:89-98.
Ferretti E, De Smaele E, Miele E, Laneve P, Po A,
Pelloni M, Paganelli A, Di Marcotullio L, Caffarelli E,
Screpanti I, Bozzoni I, Gulino A. Concerted microRNA
control of Hedgehog signalling in cerebellar neuronal
progenitor and tumour cells. EMBO J. 2008, 27:2616-27.
Ferretti E, Tosi E, Po A, Scipioni A, Morisi R,
Espinola MS, Russo D, Durante C, Schlumberger M,
Screpanti I, Filetti S, Gulino A. Notch signaling is
involved in expression of thyrocyte differentiation
markers and is downregulated in thyroid tumors. J Clin
Endocrinol Metab. 2008, 93:4080-7.