Jean-Jacques Lebrun

Activin/TGF-β induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP

Members of the transforming growth factor β (TGF-β) family regulate fundamental physiological processes, such as cell growth, differentiation and apoptosis, in almost all cell types. As a result, defects in TGF-β signalling pathways have been linked to uncontrolled cellular proliferation and carcinogenesis. Here, we explored the signal transduction mechanisms downstream of the activin/TGF-β receptors that result in cell growth arrest and apoptosis. We show that in haematopoietic cells, TGF-β family members regulate apoptosis through expression of the inositol phosphatase SHIP (Src homology 2 (SH2) domain-containing 5′ inositol phosphatase), a central regulator of phospholipid metabolism. We also demonstrated that the Smad pathway is required in the transcriptional regulation of the SHIP gene. Activin/TGF-β-induced expression of SHIP results in intracellular changes in the pool of phospholipids, as well as in inhibition of both Akt/PKB (protein kinase B) phosphorylation and cell survival. Our results link phospholipid metabolism to activin/TGF-β-mediated apoptosis and define TGF-β family members as potent inducers of SHIP expression.

PTP1D is a positive regulator of the prolactin signal leading to beta-casein promoter activation.

Stimulation of the prolactin receptor (PRLR), a member of the cytokine/growth hormone receptor family, results in activation of the associated Jak2 tyrosine kinase and downstream signaling pathways. We report that PTP1D, a cytoplasmic protein tyrosine phosphatase containing two Src homology 2 (SH2) domains, physically associates with the PRLR‐Jak2 complex and is tyrosine‐phosphorylated upon stimulation with prolactin. The formation of the trimeric PRLR‐Jak2‐PTP1D complex is critical for transmission of a lactogenic signal, while PTP1D phosphorylation is necessary, but not sufficient. The dominant negative inhibitory effect of a phosphatase‐deficient mutant on expression of a beta‐casein promoter‐controlled reporter gene is evidence for an essential role of fully functional PTP1D in the regulation of milk protein gene transcription.

The p38 MAPK Pathway Is Required for Cell Growth Inhibition of Human Breast Cancer Cells in Response to Activin

Activin, a member of the TGFβ family inhibits cell growth in various target tissues. Activin interacts with a complex of two receptors that upon activation phosphorylate specific intracellular mediators, the Smad proteins. The activated Smads interact with diverse DNA binding proteins and co-activators of transcription in a cell-specific manner, thus leading to various activin biological effects. In this study, we investigated the role and mechanism of action of activin in the human breast cancer T47D cells. We found that activin treatment of T47D cells leads to a dramatic decrease in cell growth. Thus activin appears as a potent cell growth inhibitor of these breast cancer cells. We show that activin induces the Smad pathway in these cells but also activates the p38-mitogen-activated protein kinase pathway, further leading to phosphorylation of the transcription factor ATF2. Finally, specific inhibitors of the p38 kinase (SB202190, SB203580, and PD169316) but not an inactive analogue (SB202474) or the MEK-1 inhibitor PD98059 completely abolish the activin-mediated cell growth inhibition of T47D cells. Together, these results define a new role for activin in human breast cancer T47D cells and highlight a new pathway utilized by this growth factor in the mediation of its biological effects in cell growth arrest.

The G protein‐coupled receptor kinase‐2 is a TGFβ‐inducible antagonist of TGFβ signal transduction

Signaling from the activin/transforming growth factor β (TGFβ) family of cytokines is a tightly regulated process. Disregulation of TGFβ signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G‐coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein‐coupled receptors (GPCR), as a downstream target and regulator of the TGFβ‐signaling cascade. TGFβ‐induced expression of GRK2 acts in a negative feedback loop to control TGFβ biological responses. Upon TGFβ stimulation, GRK2 associates with the receptor‐regulated Smads (R‐Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R‐Smads inhibits their carboxyl‐terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFβ‐mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFβ‐induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single‐transmembrane serine/threonine kinase receptor‐mediated signal transduction.

Activin and TGFβ regulate expression of the microRNA-181 family to promote cell migration and invasion in breast cancer cells

MicroRNA-181 (miR-181) is a multifaceted miRNA that has been implicated in many cellular processes such as cell fate determination and cellular invasion. While miR-181 is often overexpressed in human tumors, a direct role for this miRNA in breast cancer progression has not yet been characterized. In this study, we found this miRNA to be regulated by both activin and TGFβ. While we found no effect of miR-181 modulation on activin/TGFβ-mediated tumor suppression, our data clearly indicate that miR-181 plays a critical and prominent role downstream of two growth factors, in mediating their pro-migratory and pro-invasive effects in breast cancer cells miR-181 acts as a metastamir in breast cancer. Thus, our findings define a novel role for miR-181 downstream of activin/TGFβ in regulating their tumor promoting functions. Having defined miR-181 as a critical regulator of tumor progression in vitro, our results thus, highlight miR-181 as an important potential therapeutic target in breast cancer.

MicroRNA-584 and the protein phosphatase and actin regulator 1 (PHACTR1), a new signaling route through which transforming growth factor-β Mediates the migration and actin dynamics of breast cancer cells.

Background: TGF-β promotes cell migration in advanced breast cancer. Results: TGF-β down-regulates miR-584, leading to a PHACTR1 overexpression, and both are involved in cell migration and actin reorganization. Conclusion: The regulation of miR-584 and regulation of its novel target PHACTR1 are necessary steps for breast cancer cell migration. Significance: MicroRNAs offer an interesting therapeutic target in the treatment of advanced breast malignancy. TGF-β plays an important role in breast cancer progression as a prometastatic factor, notably through enhancement of cell migration. It is becoming clear that microRNAs, a new class of small regulatory molecules, also play crucial roles in mediating tumor formation and progression. We found TGF-β to down-regulate the expression of the microRNA miR-584 in breast cancer cells. Furthermore, we identified PHACTR1, an actin-binding protein, to be positively regulated by TGF-β in a miR-584-dependent manner. Moreover, we found TGF-β-mediated down-regulation of miR-584 and increased expression of PHACTR1 to be required for TGF-β-induced cell migration of breast cancer cells. Indeed, both overexpression of miR-584 and knockdown of PHACTR1 resulted in a drastic reorganization of the actin cytoskeleton and reduced TGF-β-induced cell migration. Our data highlight a novel signaling route whereby TGF-β silences the expression of miR-584, resulting in enhanced PHACTR1 expression, and further leading to actin rearrangement and breast cancer cell migration.

A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

IntroductionTumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion.MethodsA mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive.ResultsWe found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFβ/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients.ConclusionsTogether these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.

TGF-beta inhibits human cutaneous melanoma cell migration and invasion through regulation of the plasminogen activator system

Over the past decades, the incidence of cutaneous melanoma in developed countries has increased faster than any other cancer. Although most patients have localized disease at the time of diagnosis and are cured by surgical excision of the primary tumor, melanoma can be highly malignant and the survival dramatically decreases for advanced stage melanomas. It is thus necessary to understand the progression of this disease. Cell migration and invasion promote tumor metastasis, the major cause of melanoma cancer morbidity and death. In this study, we investigated the role of the TGFβ/Smad signaling pathway in melanoma tumor progression and found TGFβ to potently inhibit both cell migration and invasion in human melanoma cell lines, established from different patients. Furthermore, we elucidated the molecular mechanisms by which TGFβ exerts its effects and found the plasminogen activation system (PAS) to play a central role in the regulation of these effects. We found TGFβ to strongly up-regulate the Plasminogen Activator Inhibitor-1 (PAI-1) in melanoma cells, leading to reduced plasmin generation and activity and, in turn to inhibition of cell migration and invasion. Together, our results define TGFβ as a potent suppressor of tumor progression in cutaneous melanoma, inhibiting both cell migration and invasion.

Visible and near infrared resonance plasmonic enhanced nanosecond laser optoporation of cancer cells.

In this paper, we report a light driven, non-invasive cell membrane perforation technique based on the localized field amplification by a nanosecond pulsed laser near gold nanoparticles (AuNPs). The optoporation phenomena is investigated with pulses generated by a Nd:YAG laser for two wavelengths that are either in the visible (532 nm) or near infrared (NIR) (1064 nm). Here, the main objective is to compare on and off localized surface plasmonic resonance (LSPR) to introduce foreign material through the cell membrane using nanosecond laser pulses. The membrane permeability of human melanoma cells (MW278) has been successfully increased as shown by the intake of a fluorescent dye upon irradiation. The viability of this laser driven perforation method is evaluated by propidium iodide exclusion as well as MTT assay. Our results show that up to 25% of the cells are perforated with 532 nm pulses at 50 mJ/cm2 and around 30% of the cells are perforated with 1064 nm pulses at 1 J/cm2. With 532 nm pulses, the viability 2 h after treatment is 64% but it increases to 88% 72 h later. On the other hand, the irradiation with 1064 nm pulses leads to an improved 2 h viability of 81% and reaches 98% after 72 h. Scanning electron microscopy images show that the 5 pulses delivered during treatment induce changes in the AuNPs size distribution when irradiated by a 532 nm beam, while this distribution is barely affected when 1064 nm is used.

Activin, TGF-β and Menin in Pituitary Tumorigenesis

Pituitary adenomas are common monoclonal neoplasms accounting for approximately one-fifth of primary intracranial tumors. Prolactin-secreting pituitary adenomas (prolactinomas) are the most common form of pituitary tumors in humans. They are associated with excessive release of the hormone prolactin and increased tumor growth, giving rise to severe endocrine disorders and serious clinical concerns for the patients. Recent studies indicated that the activin/TGF-β family of growth factors plays a prominent role in regulating pituitary tumor growth and prolactin secretion from anterior pituitary lactotrope cells. Furthermore, these studies highlighted the tumor suppressor menin and the protein Smads as central regulators of these biological processes in the pituitary. Alterations in the activin/TGF-β downstream signaling pathways are critical steps towards tumor formation and progression. This chapter will review the role and intracellular molecular mechanisms of action by which activin, TGF-β, Smads and menin act in concert to prevent pituitary tumor cell growth and control hormonal synthesis by the anterior pituitary.