Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression.

Deleted in colorectal cancer (DCC), the receptor for the multifunctional cue netrin-1, acts as a tumor suppressor in intestinal cancer and lung metastasis by triggering cancer cell death when netrin-1 is lowly expressed. Recent genomic data highlighted that DCC is the third most frequently mutated gene in melanoma; we therefore investigated whether DCC could act as a melanoma tumor suppressor. Reexpressing DCC in human melanoma cell lines promoted tumor cell death and tumor growth inhibition in xenograft mouse models. Genetic silencing of DCC prodeath activity in a BRAFV600E mouse model increased the proportion of mice with melanoma, further supporting that DCC is a melanoma tumor suppressor. Netrin-1 expression was elevated in melanoma compared with benign melanocytic lesions. Upregulation of netrin-1 in the skin cells of a BRAFV600E-mutated murine model reduced cancer cell death and promoted melanoma progression. Therapeutic antibody blockade of netrin-1 combined with dacarbazine increased overall survival in several mouse melanoma models. Together, these data support that interfering with netrin-1 could be a viable therapeutic approach in patients with netrin-1-expressing melanoma. SIGNIFICANCE: Netrin-1 and its receptor DCC regulate melanoma progression, suggesting therapeutic targeting of this signaling axis as a viable option for melanoma treatment.

The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor.

c-Kit is a classic proto-oncogene either mutated or upregulated in cancer cells, and this leads to its constitutive kinase activation and, thus, to uncontrolled proliferation. Although the pro-oncogenic role of c-Kit is of no doubt, some observations do not fit well with c-Kit solely as a tumor-promoting moiety. We show here that c-Kit actively triggers cell death in various cancer cell lines unless engaged by its ligand stem cell factor (SCF). This pro-death activity is enhanced when the kinase activation of c-Kit is silenced and is due to c-Kit intracellular cleavage by caspase-like protease at D816. Moreover, in vivo, overexpression of a c-Kit kinase-dead mutant inhibits tumor growth, and this intrinsic c-Kit tumor-suppressive activity is dependent on the D816 cleavage. Thus, c-Kit acts both as a proto-oncogene via its kinase activity and as a tumor suppressor via its dependence receptor activity.

Structural Decoding of the Netrin-1/UNC5 Interaction and its Therapeutical Implications in Cancers.

Netrin-1 has been shown to be up-regulated in a fraction of human cancers as a mechanism to allow these tumors to escape the pro-apoptotic activity of some of its main dependence receptors, the UNC5 homologs (UNC5H). Here we identify the V-2 domain of netrin-1 to be important for its interaction with the Ig1/Ig2 domains of UNC5H2. We generate a humanized anti-netrin-1 antibody that disrupts the interaction between netrin-1 and UNC5H2 and triggers death of netrin-1-expressing tumor cells in vitro. We also present evidence that combining the anti-netrin-1 antibody with epidrugs such as decitabine could be effective in treating tumors showing no or modest netrin-1 expression. These results support that this antibody is a promising drug candidate.

The thyroid hormone nuclear receptor TRα1 controls the Notch signaling pathway and cell fate in murine intestine.

Thyroid hormones control various aspects of gut development and homeostasis. The best-known example is in gastrointestinal tract remodeling during amphibian metamorphosis. It is well documented that these hormones act via the TR nuclear receptors, which are hormone-modulated transcription factors. Several studies have shown that thyroid hormones regulate the expression of several genes in the Notch signaling pathway, indicating a possible means by which they participate in the control of gut physiology. However, the mechanisms and biological significance of this control have remained unexplored. Using multiple in vivo and in vitro approaches, we show that thyroid hormones positively regulate Notch activity through the TRα1 receptor. From a molecular point of view, TRα1 indirectly controls Notch1, Dll1, Dll4 and Hes1 expression but acts as a direct transcriptional regulator of the Jag1 gene by binding to a responsive element in the Jag1 promoter. Our findings show that the TRα1 nuclear receptor plays a key role in intestinal crypt progenitor/stem cell biology by controlling the Notch pathway and hence the balance between cell proliferation and cell differentiation.

Acacetin and chrysin, two polyphenolic compounds, alleviate telomeric position effect in human cells.

We took advantage of the ability of human telomeres to silence neighboring genes (telomere position effect or TPE) to design a high-throughput screening assay for drugs altering telomeres. We identified, for the first time, that two dietary flavones, acacetin and chrysin, are able to specifically alleviate TPE in human cells. We further investigated their influence on telomere integrity and showed that both drugs drastically deprotect telomeres against DNA damage response. However, telomere deprotection triggered by shelterin dysfunction does not affect TPE, indicating that acacetin and chrysin target several functions of telomeres. These results show that TPE-based screening assays represent valuable methods to discover new compounds targeting telomeres.Molecular Therapy-Nucleic Acids (2013) 2, e116; doi:10.1038/mtna.2013.42; published online 20 August 2013.

Replication timing of human telomeres is chromosome arm-specific, influenced by subtelomeric structures and connected to nuclear localization.

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of beta-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4-associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm-specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.

Identification of a perinuclear positioning element in human subtelomeres that requires A-type lamins and CTCF.

The localization of genes within the nuclear space is of paramount importance for proper genome functions. However, very little is known on the cis-acting elements determining subnuclear positioning of chromosome segments. We show here that the D4Z4 human subtelomeric repeat localizes a telomere at the nuclear periphery. This perinuclear activity lies within an 80 bp sequence included within a region known to interact with CTCF and A-type Lamins. We further show that a reduced level of either CTCF or A-type Lamins suppresses the perinuclear activities of D4Z4 and that an array of multimerized D4Z4 sequence, which has lost its ability to bind CTCF and A-type Lamins, is not localized at the periphery. Overall, these findings reveal the existence of an 80 bp D4Z4 sequence that is sufficient to position an adjacent telomere to the nuclear periphery in a CTCF and A-type lamins-dependent manner. Strikingly, this sequence includes a 30 bp GA-rich motif, which binds CTCF and is present at several locations in the human genome.

The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy.

Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD), which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s) through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer-promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients.