Alain Puisieux

Regulatory T Cells Recruited through CCL22/CCR4 Are Selectively Activated in Lymphoid Infiltrates Surrounding Primary Breast Tumors and Lead to an Adverse Clinical Outcome

Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3(+) T cells are typical Treg based on their CD4(+)CD25(high)CD127(low)FOXP3(+) phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP(+) dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3(+), and CD8(+) T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem.

p53 mutation in hepatocellular carcinoma after aflatoxin exposure

Mutations of the p53 gene are found in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Specific mutations might reflect exposure to specific carcinogens and we have screened HCC samples from patients in 14 different countries to determine the frequency of a hotspot mutation at codon 249 of the tumour suppressor p53 gene. We detected mutations in 17% of tumours (12/72) from four countries in south Africa and the southeast coast of Asia. There was no codon 249 mutation in 95 specimens of HCC from other geographical locations including North America, Europe, Middle East, and Japan. Worldwide, the presence of the codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus (HBV). Further studies were completed in two groups of HBV-infected patients at different risks of exposure to aflatoxins. 53% of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumour with a codon 249 mutation, in contrast with 8% of patients from Transkei (1/12) who were at low risk. HCC is an endemic disease in Mozambique and accounts for up to two thirds of all tumours in men. A codon 249 mutation of the p53 gene identifies an endemic form of HCC strongly associated with dietary aflatoxin intake.

Oncogenic roles of EMT-inducing transcription factors.

The plasticity of cancer cells underlies their capacity to adapt to the selective pressures they encounter during tumour development. Aberrant reactivation of epithelial–mesenchymal transition (EMT), an essential embryonic process, can promote cancer cell plasticity and fuel both tumour initiation and metastatic spread. Here we discuss the roles of EMT-inducing transcription factors in creating a pro-tumorigenic setting characterized by an intrinsic ability to withstand oncogenic insults through the mitigation of p53-dependent oncosuppressive functions and the gain of stemness-related properties.

Impaired IFN-α Production by Plasmacytoid Dendritic Cells Favors Regulatory T-cell Expansion That May Contribute to Breast Cancer Progression

Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3(+) Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer.

Alterations of anaphase-promoting complex genes in human colon cancer cells

Ubiquitin-mediated proteolysis of cell cycle regulators is a major element of the cell cycle control. The anaphase-promoting complex (APC/C) is a large multisubunit ubiquitin-protein ligase required for the ubiquitination and degradation of G1 and mitotic checkpoint regulators. APC/C-dependent proteolysis regulates cyclin levels in G1, and triggers the separation of sister chromatids at the metaphase–anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Furthermore, it was recently shown that APC/C regulates the degradation of crucial regulators of signal transduction pathways. We report here gene alterations in several components of this complex in human colon cancer cells, including APC6/CDC16 and APC8/CDC23 which are known to be key function elements. The experimental expression of a truncation mutant of APC8/CDC23 subunit (CDC23ΔTPR) leads to abnormal levels of APC/C targets such as cyclin B1 and disturbs the cell cycle progression of colon epithelial cells through mitosis. Overall, these data support the hypothesis of a deleterious role of these mutations during colorectal carcinogenesis.

BTG2(TIS21/PC3) induces neuronal differentiation and prevents apoptosis of terminally differentiated PC12 cells.

The p53-transcriptional target, BTG2TIS21/PC3, was previously identified as an antiproliferative gene. However, the precise biological functions of the protein product remain to be elucidated. BTG2TIS21/PC3 expression is induced in vivo during neurogenesis, and the gene is transiently expressed in vitro in rat pheochromocytoma PC12 cells after induction of neuronal differentiation by addition of nerve growth factor (NGF). These observations suggest that BTG2TIS21/PC3 is functionally significant during the neuronal differentiation process. To test this hypothesis, a vector that expressed BTG2TIS21/PC3 under the control of an inducible promoter was introduced into PC12 cells. Growth arrest and differentiation in response to NGF were greatly enhanced by BTG2TIS21/PC3 overexpression. Furthermore, an antisense oligonucleotide complementary to BTG2TIS21/PC3 mRNA, which was able to inhibit endogenous BTG2TIS21/PC3 expression, triggered programmed cell death in differentiated PC12 cells. These observations confirm that BTG2TIS21/PC3 expression promotes neuronal differentiation and that it is required for survival of terminally differentiated cells.

Cancer Stem Cells: The Emerging Challenge of Drug Targeting

Stem cells are defined by their unique property to self-renew and starting from one single cell to generate all the different progenies required for tissue regeneration. In adults, stem cells are still present in the majority of tissues and organs where they are responsible for continuous organs and tissues homeostasis. Adult stem cells have been isolated in various tissues and all share common specific characteristics (localization in stem cell niches, drug transporter expression, adhesion, levels of apoptosis inhibitors, DNA methylation, -) involved in high levels of drug resistance of this specific cell subtype. Several studies have identified different populations of cancer cells, within the same tumor, some of them which present properties closely related to normal stem cells and raised the concept of cancer stem cells. Interestingly, the cell surface markers expressed by these particular cancer cells are the same as those expressed by normal stem cells, suggesting that cancer can arise in some cases from the malignant transformation of stem cells. The cancer stem cell (CaSC) model predicts that, even if conventional cancer cells can be killed by chemotherapy or radiotherapy, only the destruction of CaSC, considered responsible for relapse, will allow full recovery, thus demonstrating the importance of CaSC-targeting for patient outcome. Therapeutic innovations will emerge from a better understanding of the biology and environment of cancer stem cells. The tumor environment can create a niche favoring the survival and proliferation of CaSC. It also protects CaSC from chemotherapy-induced apoptosis. Clinically, it is crucial to get rid of quiescent and resistant cells and to adapt the therapeutic strategy to cancer stem cells sheltered in niches. Here, we review the major characteristics of cancer stem cells and their behavior in response to chemotherapy; we also highlight the main issues to be considered for efficient and specific cancer stem cell targeting.

The CD10 enzyme is a key player to identify and regulate human mammary stem cells.

The major components of the mammary ductal tree are an inner layer of luminal cells, an outer layer of myoepithelial cells, and a basement membrane that separates the ducts from the underlying stroma. Cells in the outer layer express CD10, a zinc‐dependent metalloprotease that regulates the growth of the ductal tree during mammary gland development. To define the steps in the human mammary lineage at which CD10 acts, we have developed an in vitro assay for human mammary lineage progression. We show that sorting for CD10 and EpCAM cleanly separates progenitors from differentiated luminal cells and that the CD10‐high EpCAM‐low population is enriched for early common progenitor and mammosphere‐forming cells. We also show that sorting for CD10 enriches sphere‐forming cells from other tissue types, suggesting that it may provide a simple tool to identify stem or progenitor populations in tissues for which lineage studies are not currently possible. We demonstrate that the protease activity of CD10 and the adhesion function of β1‐integrin are required to prevent differentiation of mammary progenitors. Taken together, our data suggest that integrin‐mediated contact with the basement membrane and cleavage of signaling factors by CD10 are key elements in the niche that maintains the progenitor and stem cell pools in the mammary lineage. STEM Cells 2010;28:1081–1088

BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling

Activin A, BMP2, and BMP4, 3 members of the transforming growth factor-beta family, are involved in the regulation of hematopoiesis. Here, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay. Our results highlight for the first time that, in the absence of thrombopoietin, BMP4 is able to induce CD34(+) progenitor differentiation into megakaryocytes through all stages. Although we have previously shown that activin A and BMP2 are involved in erythropoietic commitment, these molecules have no effect on human megakaryopoietic engagement and differentiation. Using signaling pathway-specific inhibitors, we show that BMP4, like thrombopoietin, exerts its effects on human megakaryopoiesis through the JAK/STAT and mTor pathways. Inhibition of the BMP signaling pathway with blocking antibodies, natural soluble inhibitors (FLRG or follistatin), or soluble BMP receptors reveals that thrombopoietin uses the BMP4 pathway to induce megakaryopoiesis, whereas the inverse is not occurring. Finally, we show that thrombopoietin up-regulates the BMP4 autocrine loop in megakaryocytic progenitors by inducing their production of BMP4 and up-regulating BMP receptor expression. In summary, this work indicates that BMP4 plays an important role in the control of human megakaryopoiesis.

p21(WAF1/CIP1) response to genotoxic agents in wild-type TP53 expressing breast primary tumours.

Functional inactivation of the wild-type p53 protein has been described in different human cancers. Since a significant proportion of breast tumours express wild-type TP53, the p53 antiproliferative activity could be inactivated in transformed mammary epithelial cells by a mechanism independent on structural alteration of the gene. To test this hypothesis, we analysed the p53 activity in primary breast tumour cells. As a preliminary study, we demonstrated in breast adenocarcinoma cell lines that the nuclear accumulation of the inhibitor of cyclin dependent kinase p21WAF1/CIP1, in response to adriamycin treatment, specifically reflected the activity of a functional wild-type p53 protein. Then, we used this strategy to study the p53 activity in 23 primary breast tumours. p21WAF1/CIP1 accumulation was detected in all tumours expressing wild-type TP53. In contrast, no p21WAF1/CIP1 response was detected in cells harboring a mutant TP53 gene. This report is the first functional study of p53 in primary breast tumours. The results demonstrate that TP53 mutation represents the only common mechanism leading to an irreversible inactivation of p53 functions in this cancer type.