François Lallemand

Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling.

Members of the TGF-β family of growth factors signal from the cell surface through serine/threonine kinase receptors. Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family. Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-β signaling. A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein. The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells. Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains. We demonstrate that YAP65 potentiates the inhibitory activity of Smad7 against TGF-β-induced, Smad3/4-dependent, gene transactivation. Furthermore, YAP65 augments the association of Smad7 to activated TGF-β receptor type I (TβRI), whereas YAP65(1–301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-β signaling, reduces these interactions. Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TβRI, and enhances the inhibitory activity of Smad7 against TGF-β signaling.

Smad7 inhibits the survival nuclear factor κB and potentiates apoptosis in epithelial cells

In this study, we examined the effect of the stable expression of Smad7 in two different cell lines on apoptosis induced by various stimuli including TGF-β, serum withdrawal, loss of cell adhesion (anoikis) and TNF-α. Smad7 increased TGF-β-mediated apoptosis in Mv1Lu cells as well as anoikis and/or serum withdrawal-induced apoptosis in Mv1Lu and MDCK cells. Smad7 markedly decreased the activity of the survival NF-κB transcription factor in MDCK cells. Interestingly, the stable expression of oncogenic Ras in MDCK cells which suppressed Smad7 inhibition of NF-κB also suppressed Smad7 potentiation of serum withdrawal-induced apoptosis and anoikis. In addition, Smad7 inhibited TNF-α stimulation of NF-κB and increased TNF-α-mediated apoptosis in MDCK cells. Our results provide the first evidence that Smad7 induces sensitization of cells to different forms of cell death. They moreover demonstrate that Smad7 inhibits the survival NF-κB factor, providing a potential mechanism whereby Smad7 potentiates cell death.

c-Jun interacts with the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity.

The Sma and Mad related (Smad) family proteins are critical mediators of the transforming growth factor-β (TGF-β) superfamily signaling. After TGF-β-mediated phosphorylation and association with Smad4, Smad2 moves to the nucleus and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of transcription factors, forkhead activin signal transducer (FAST)-1 and FAST2. TGF-β has also been described to activate other signaling pathways, such as the c-Jun N-terminal Kinase (JNK) pathway. Here, we show that activation of JNK cascade blocked the ability of Smad2 to mediate TGF-β-dependent activation of the FAST proteins. This inhibitory activity is mediated through the transcriptional factor c-Jun, which enhances the association of Smad2 with the nuclear transcriptional corepressor TG-interacting factor (TGIF), thereby interfering with the assembly of Smad2 and the coactivator p300 in response to TGF-β signaling. Interestingly, c-Jun directly binds to the nuclear transcriptional corepressor TGIF and is required for TGIF-mediated repression of Smad2 transcriptional activity. These studies thus reveal a mechanism for suppression of Smad2 signaling pathway by JNK cascade through transcriptional repression.

AIP4 Restricts Transforming Growth Factor-β Signaling through a Ubiquitination-independent Mechanism

Smad7 functions as an intracellular antagonist in transforming growth factor-β (TGF-β) signaling. In addition to interacting stably with the activated TGF-β type I receptor (TβRI) to prevent phosphorylation of the receptor-regulated Smads (Smad2 and Smad3), Smad7 also induces degradation of the activated TβRI through association with different E3 ubiquitin ligases. Using the two-hybrid screen, we identified atrophin 1-interacting protein 4 (AIP4) as an E3 ubiquitin ligase that specifically targets Smad7 for ubiquitin-dependent degradation without affecting the turnover of the activated TβRI. Surprisingly, we found that despite the ability to degrade Smad7, AIP4 can inhibit TGF-β signaling, presumably by enhancing the association of Smad7 with the activated TβRI. Consistent with this notion, expression of a catalytic mutant of AIP4, which is unable to induce ubiquitination and degradation of Smad7, also stabilizes the TβRI·Smad7 complex, resulting in inhibition of TGF-β signaling. The ability of AIP4 to enhance the inhibitory function of Smad7 independent of its ubiquitin ligase activity reveals a new mechanism by which E3 ubiquitin ligases may function to turn off TGF-β signaling.

The Wnt Antagonist Dickkopf-1 Increases Endothelial Progenitor Cell Angiogenic Potential

Objective—To determine the role of Wnt antagonist Dickkopf (DKK) 1 in human endothelial colony-forming cells (ECFCs) in view of the emerging importance of Wnt pathways in vascular biology. Methods and Results—Endothelial progenitor cells have been proposed to be crucial in tumor neovascularization. Recombinant DKK1 has been tested in ECFC angiogenic properties in vitro. DKK1 enhanced ECFC proliferation and the capacity of ECFCs to form pseudotubes in Matrigel. These effects have been attributed to enhancement of vascular endothelial growth factor receptor 2, SDF-1, and CXCR4. DKK1 gene silencing has been realized on ECFCs and mesenchymal stem cells, and we found that DKK1 silencing in the 2 cell types decreased their angiogenic potential. We then examined the possible role of DKK1 in tumor neovasculogenesis and found that blood vessels of breast cancer tissues expressed DKK1 far more strongly in human breast tumors than in normal breast tissues. By studying 62 human breast tumors, we found a significant positive correlation between DKK1 expression and von Willebrand factor. In vivo, DKK1 strongly enhanced the vascularization of Matrigel plugs and increased tumor size in a xenograft model of human breast carcinoma in nude mice. Conclusion—DKK1 enhances angiogenic properties of ECFCs in vitro and is required for ECFC and mesenchymal stem cell angiogenic phenotypes in vivo. DKK1 also increases tumoral angiogenesis. Thus, we demonstrated a major role of DKK1 in angiogenic processes.

Role of the Focal Adhesion Protein Kindlin-1 in Breast Cancer Growth and Lung Metastasis

BACKGROUND Fermitin family member 1 (FERMT1, Kindlin-1) is an epithelial-specific regulator of integrin functions and is associated with Kindler syndrome, a genetic disorder characterized by skin blistering, atrophy, and photosensitivity. However, the possible role of kindlin-1 in cancer remains unknown. METHODS Kindlin-1 expression was quantified in several human cancers using quantitative real-time polymerase chain reaction and published microarray datasets. The association between kindlin-1 expression and patient metastasis-free survival (N = 516) was assessed with Kaplan-Meier analyses. Effects of ectopic expression or silencing of kindlin-1 on cell signaling, migration, and invasion were assessed in human breast cancer cell lines using western blotting, immunofluorescence, wound healing assays, and invasion on Matrigel or type I collagen substrates. Breast tumor growth and lung metastasis were evaluated in 12-week-old female BALB/c mice (10 controls and six Kindlin-1-knockdown mice). All statistical tests were two-sided. RESULTS Kindlin-1 expression was consistently higher in tumors than in normal tissues in various cancer types metastasizing to the lungs, including colon and bladder cancer. Kindlin-1 expression was associated with metastasis-free survival in both breast and lung adenocarcinoma (breast cancer: hazard ratio of lung metastasis = 2.55, 95% confidence intervals [CI] = 1.39 to 4.69, P = .001; lung cancer: hazard ratio of metastasis = 1.96, 95% CI = 1.25 to 3.07, P = .001). Overexpression of kindlin-1 induced changes indicating epithelial-mesenchymal transition and transforming growth factor beta (TGFβ) signaling, constitutive activation of cell motility, and invasion (number of migrating cells, Kindlin-1 cells vs control, mean = 164.66 vs. 19.00, difference = 145.6, 95% CI = 79.1 to 212.2, P = .004; invasion rate, Kindlin-1-cells vs control = 9.65% vs. 1.92%, difference = 7.73%, 95% CI = 4.75 to 10.70, P < .001). Finally, Kindlin-1 depletion in an orthotopic mouse model statistically significantly inhibited breast tumor growth (P < .001) and lung metastasis (P = .003). CONCLUSION These results suggest a role for kindlin-1 in breast cancer lung metastasis and lung tumorigenesis and advance our understanding of kindlin-1 as a regulator of TGFβ signaling, offering new avenues for therapeutic intervention against cancer progression.

Expression analysis of mitotic spindle checkpoint genes in breast carcinoma: role of NDC80/HEC1 in early breast tumorigenicity, and a two-gene signature for aneuploidy

BackgroundAneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes, but little is known about somatic alterations of mitotic spindle checkpoint genes in breast cancer.MethodsTo obtain further insight into the molecular mechanisms underlying aneuploidy in breast cancer, we used real-time quantitative RT-PCR to quantify the mRNA expression of 76 selected mitotic spindle checkpoint genes in a large panel of breast tumor samples.ResultsThe expression of 49 (64.5%) of the 76 genes was significantly dysregulated in breast tumors compared to normal breast tissues: 40 genes were upregulated and 9 were downregulated. Most of these changes in gene expression during malignant transformation were observed in epithelial cells.Alterations of nine of these genes, and particularly NDC80, were also detected in benign breast tumors, indicating that they may be involved in pre-neoplastic processes.We also identified a two-gene expression signature (PLK1 + AURKA) which discriminated between DNA aneuploid and DNA diploid breast tumor samples. Interestingly, some DNA tetraploid tumor samples failed to cluster with DNA aneuploid breast tumors.ConclusionThis study confirms the importance of previously characterized genes and identifies novel candidate genes that could be activated for aneuploidy to occur. Further functional analyses are required to clearly confirm the role of these new identified genes in the molecular mechanisms involved in breast cancer aneuploidy. The novel genes identified here, and/or the two-gene expression signature, might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies.

The Activation of the WNT Signaling Pathway Is a Hallmark in Neurofibromatosis Type 1 Tumorigenesis

Purpose: The hallmark of neurofibromatosis type 1 (NF1) is the onset of dermal or plexiform neurofibromas, mainly composed of Schwann cells. Plexiform neurofibromas can transform into malignant peripheral nerve sheath tumors (MPNST) that are resistant to therapies. Experimental Design: The aim of this study was to identify an additional pathway in the NF1 tumorigenesis. We focused our work on Wnt signaling that is highly implicated in cancer, mainly in regulating the proliferation of cancer stem cells. We quantified mRNAs of 89 Wnt pathway genes in 57 NF1-associated tumors including dermal and plexiform neurofibromas and MPNSTs. Expression of two major stem cell marker genes and five major epithelial–mesenchymal transition marker genes was also assessed. The expression of significantly deregulated Wnt genes was then studied in normal human Schwann cells, fibroblasts, endothelial cells, and mast cells and in seven MPNST cell lines. Results: The expression of nine Wnt genes was significantly deregulated in plexiform neurofibromas in comparison with dermal neurofibromas. Twenty Wnt genes showed altered expression in MPNST biopsies and cell lines. Immunohistochemical studies confirmed the Wnt pathway activation in NF1-associated MPNSTs. We then confirmed that the knockdown of NF1 in Schwann cells but not in epithelial cells provoked the activation of Wnt pathway by functional transfection assays. Furthermore, we showed that the protein expression of active β-catenin was increased in NF1-silenced cell lines. Wnt pathway activation was strongly associated to both cancer stem cell reservoir and Schwann–mesenchymal transition. Conclusion: We highlighted the implication of Wnt pathway in NF1-associated tumorigenesis. Clin Cancer Res; 20(2); 358–71. ©2013 AACR.

Prognostic value of a newly identified MALAT1 alternatively spliced transcript in breast cancer

Background:Epigenetic deregulation is considered as a new hallmark of cancer. The long non-coding RNA MALAT1 has been implicated in several cancers; however, its role in breast cancer is still little known.Methods:We used RT–PCR, in situ hybridisation, and RPPA methods to quantify (i) the full-length (FL) and an alternatively spliced variant (Δsv) of MALAT1, and (ii) a panel of transcripts and proteins involved in MALAT1 pathways, in a large series of breast tumours from patients with known clinical/pathological status and long-term outcome.Results:MALAT1 was overexpressed in 14% (63/446) of the breast tumours. MALAT1-overexpressed tumour epithelial cells showed marked diffuse nuclear signals and numerous huge nuclear speckles. Screening of the dbEST database led to the identification of Δsv-MALAT1, a major alternatively spliced MALAT1 transcript, with a very different expression pattern compared with FL-MALAT1. This alternative Δsv-MALAT1 transcript was mainly underexpressed (18.8%) in our breast tumour series. Multivariate analysis showed that alternative Δsv-MALAT1 transcript is an independent prognostic factor. Δsv-MALAT1 expression was associated with alterations of the pre-mRNAs alternative splicing machinery, and of the Drosha-DGCR8 complex required for non-coding RNA biogenesis. Alternative Δsv-MALAT1 transcript expression was associated to YAP protein status and with an activation of the PI3K-AKT pathway.Conclusions:Our results reveal a complex expression pattern of various MALAT1 transcript variants in breast tumours, and suggest that this pattern of expressions should be taken into account to evaluate MALAT1 as predictive biomarker and therapeutic target.

MiR-190b, the highest up-regulated miRNA in ERα-positive compared to ERα-negative breast tumors, a new biomarker in breast cancers?

BackgroundMicroRNAs (miRNAs) show differential expression across breast cancer subtypes and have both oncogenic and tumor-suppressive roles. Numerous microarray studies reported different expression patterns of miRNAs in breast cancers and found clinical interest for several miRNAs but often with contradictory results. Aim of this study is to identify miRNAs that are differentially expressed in estrogen receptor positive (ER+) and negative (ER−) breast primary tumors to better understand the molecular basis for the phenotypic differences between these two sub-types of carcinomas and to find potential clinically relevant miRNAs.MethodsWe used the robust and reproductive tool of quantitative RT-PCR in a large cohort of well-annotated 153 breast cancers with long-term follow-up to identify miRNAs specifically differentially expressed between ER+ and ER− breast cancers. Cytotoxicity tests and transfection experiments were then used to examine the role and the regulation mechanisms of selected miRNAs.ResultsWe identified a robust collection of 20 miRNAs significantly deregulated in ER+ compared to ER− breast cancers : 12 up-regulated and eight down-regulated miRNAs. MiR-190b retained our attention as it was the miRNA the most strongly over-expressed in ER+ compared to ER− with a fold change upper to 23. It was also significantly up-regulated in ER+/Normal breast tissue and down-regulated in ER−/Normal breast tissue. Functional experiments showed that miR-190b expression is not directly regulated by estradiol and that miR-190b does not affect breast cancer cell lines proliferation. Expression level of miR-190b impacts metastasis-free and event-free survival independently of ER status.ConclusionsThis study reveals miR-190b as the highest up-regulated miRNA in hormone-dependent breast cancers. Due to its specificity and high expression level, miR-190b could therefore represent a new biomarker in hormone-dependent breast cancers but its exact role carcinogenesis remains to elucidate.