Proper regulation of white and brown adipogenic differentiation is important for maintaining an organism's metabolic profile in a homeostatic state. The recent observations showing that the p53 tumor suppressor plays a role in metabolism raise the question of whether it is involved in the regulation of white and brown adipocyte differentiation. By using several in vitro models, representing various stages of white adipocyte differentiation, we found that p53 exerts a suppressive effect on white adipocyte differentiation in both mouse and human cells. Moreover, our in vivo analysis indicated that p53 is implicated in protection against diet-induced obesity. In striking contrast, our data shows that p53 exerts a positive regulatory effect on brown adipocyte differentiation. Abrogation of p53 function in skeletal muscle committed cells reduced their capacity to differentiate into brown adipocytes and histological analysis of brown adipose tissue revealed an impaired morphology in both embryonic and adult p53-null mice. Thus, depending on the specific adipogenic differentiation program, p53 may exert a positive or a negative effect. This cell type dependent regulation reflects an additional modality of p53 in maintaining a homeostatic state, not only in the cell, but also in the organism at large.

Microenvironmental factors contribute to the immune dysfunction characterizing acute myeloid leukemia (AML). Indoleamine 2,3-dioxygenase 1 (IDO1) is an interferon (IFN)-γ-inducible enzyme that degrades tryptophan into kynurenine, which, in turn, inhibits effector T cells and promotes regulatory T-cell (Treg) differentiation. It is presently unknown whether childhood AML cells express IDO1 and whether IDO1 activity correlates with patient outcome.We investigated IDO1 expression and function in 37 children with newly diagnosed AML other than acute promyelocytic leukemia. Blast cells were cultured with exogenous IFN-γ for 24 hours, followed by the measurement of kynurenine production and tryptophan consumption. No constitutive expression of IDO1 protein was detected in blast cells from the 37 AML samples herein tested. Conversely, 19 out of 37 (51%) AML samples up-regulated functional IDO1 protein in response to IFN-γ. The inability to express IDO1 by the remaining 18 AML samples was not apparently due to a defective IFN-γ signaling circuitry, as suggested by the measurement of signal transducer and activator of transcription 3 (STAT3) phosphorylation. Co-immunoprecipitation assays indicated the occurrence of physical interactions between STAT3 and IDO1 in AML blasts. In line with this finding, STAT3 inhibitors abrogated IDO1 function in AML blasts. Interestingly, levels of IFN-γ were significantly higher in the bone marrow fluid of IDO-expressing compared with IDO-nonexpressing AMLs. In mixed tumor lymphocyte cultures (MTLC), IDO-expressing AML blasts blunted the ability of allogeneic naïve T cells to produce IFN-γ and promoted Treg differentiation. From a clinical perspective, the 8-year event-free survival was significantly worse in IDO-expressing children (16.4%, SE 9.8) as compared with IDO-nonexpressing ones (48.0%, SE 12.1; p=0.035).These data indicate that IDO1 expression by leukemia blasts negatively affects the prognosis of childhood AML. Moreover, they speak in favor of the hypothesis that IDO can be targeted, in adjunct to current chemotherapy approaches, to improve the clinical outcome of children with AML.

The integrin A 6 B 4 has been shown to facilitate key functions of carcinoma cells, including their ability to migrate, invade, and evade apoptosis. The mechanism involved seems to be a profound effect of A 6 B 4 on specific signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An intimate relationship between A 6 B 4 and growth factor receptors may explain this effect of A 6 B 4 on signaling. Previously, we showed that A 6 B 4 and ErbB-2 can function synergistically to activate the PI3K/Akt pathway. Given that ErbB-2 can activate PI3K only when it heterodimerizes with other members of the epidermal growth factor receptor family, these data imply that other receptors cooperate in this process. Here, we report that A 6 B 4 can regulate the expression of ErbB-3 using several different models and that the consequent formation of an ErbB-2/ErbB-3 heterodimer promotes the A 6 B 4 -dependent activation of PI3K/Akt and the ability of this integrin to impede apoptosis of carcinoma cells. Our data also support the hypothesis that A 6 B 4 can regulate ErbB-3 expression at the translational level as evidenced by the findings that A 6 B 4 does not increase ErbB-3 mRNA significantly, and that this regulation is both rapamycin sensitive and dependent on eukaryotic translation initiation factor 4E. These findings provide one mechanism to account for the activation of PI3K by A 6 B 4 and they also provide insight into the regulation of ErbB-3 in carcinoma cells. [Cancer Res 2007;67(4):1645-52]

BackgroundMultiple myeloma (MM) is a plasma cell malignancy with a multifaceted immune dysfunction. Indoleamine 2,3-dioxygenase 1 (IDO1) degrades tryptophan into kynurenine (KYN), which inhibits effector T cells and promote regulatory T-cell (Treg) differentiation. It is presently unknown whether MM cells express IDO1 and whether IDO1 activity correlates with immune system impairment.MethodsWe investigated IDO1 expression in 25 consecutive patients with symptomatic MM and in 7 patients with either monoclonal gammopathy of unknown significance (MGUS; n=3) or smoldering MM (SMM; n=4). IDO1-driven tryptophan breakdown was correlated with the release of hepatocyte growth factor (HGF) and with the frequency of Treg cells and NY-ESO-1-specific CD8+ T cells.ResultsKYN was increased in 75% of patients with symptomatic MM and correlated with the expansion of CD4+CD25+FoxP3+ Treg cells and the contraction of NY-ESO-1-specific CD8+ T cells. In vitro, primary MM cells promoted the differentiation of allogeneic CD4+ T cells into bona fide CD4+CD25hiFoxP3hi Treg cells and suppressed IFN-γ/IL-2 secretion, while preserving IL-4 and IL-10 production. Both Treg expansion and inhibition of Th1 differentiation by MM cells were reverted, at least in part, by d,l-1-methyl-tryptophan, a chemical inhibitor of IDO. Notably, HGF levels were higher within the BM microenvironment of patients with IDO+ myeloma disease compared with patients having IDO- MM. Mechanistically, the antagonism of MET receptor for HGF with SU11274, a MET inhibitor, prevented HGF-induced AKT phosphorylation in MM cells and translated into reduced IDO protein levels and functional activity.ConclusionsThese data suggest that IDO1 expression may contribute to immune suppression in patients with MM and possibly other HGF-producing cancers.

Purpose: The a 6 h 4 integrin, a laminin receptor, has been implicated from many studies in tumor progression and invasion. We showed that the h 4 integrin subunit associates with the ErbB-2 tyrosine kinase in human mammary carcinoma cell lines and that its overexpression in NIH3T3/ ErbB-2^transformed cells causes a constitutive activation of phosphatidylinositol 3-kinase (PI3K), inducing a strong increase of their invasive capacity. In this study, we investigated the biological consequences of interference with the endogenous h 4 integrin subunit expression. Experimental Design: In vitro and in vivo tumor growth and the biochemical consequences of h 4 integrin inactivation were studied in mammary tumor cells by using short hairpin RNA approach. Results: Our data show that tumor growth of mammary tumor cells strictly depends on h 4 expression, confirming the relevance of h 4 protein in these cells. Moreover, interference with h 4 expression significantly reduces endogenous PI3K activity and AKT and mammalian target of rapamycin phosphorylation. Accordingly, with these results and considering that PI3K activity in mammary tumor plays a relevant role in hormone resistance, we asked whether h 4 expression might be relevant for hormone responsiveness in these cells. Data reported indicate that the interference with endogenous h 4 expression, upon hormone deprivation, induces caspase-9 and cytochrome c^mediated apoptosis, which is enhanced upon tamoxifen treatment. On the other hand, the expression of myr-AKT in MCF7 h 4^s hort hairpin RNA cells rescues the cells from apoptosis in the absence of hormones and upon tamoxifen treatment. Conclusions: Overall, these results confirm the relevance of h 4 expression in mammary tumors and indicate this integrin as a relevant target for tumor therapy.

BackgroundTamoxifen is still the most widely used drug in hormone therapy for the treatment of breast cancer. Its benefits in adjuvant treatment are well documented in controlled and randomized clinical studies, which have demonstrated an increase in disease-free intervals of patients with positive hormonal receptors. However, the mechanisms involved in endocrine resistance are not clear. Laboratory and clinical data now indicate that bi-directional molecular cross-talk between nuclear or membrane ER and growth factor receptor pathways may be involved in endocrine resistance. We recently found a functional interaction between α6β4 integrin and ErbB-3 receptor to maintain the PI3K/Akt survival pathway of mammary tumour cells. We sought to improve understanding of this process in order to provide the involvement of both receptors insight into mechanism of Tamoxifen resistance.Methods and FindingsUsing human breast cancer cell lines displaying different levels of α6β4 and ErbB-3 receptors and a series of 232 breast cancer biopsies from patients submitted to adjuvant Tamoxifen monotherapy for five years, we evaluated the functional interaction between both receptors in relationship to Tamoxifen responsiveness. In mammary carcinoma cells, we evidenced that the α6β4 integrin strongly influence Akt phosphorylation through ErbB-3 protein regulation. Moreover, the ErbB-3 inactivation inhibits Akt phosphorylation, induces apoptosis and inhibits in vitro invasion favouring Tamoxifen responsiveness. The analysis of human tumors revealed a significant relationship between α6β4 and ErbB-3 in P-Akt-positive and ERβ1-negative breast cancers derived from patients with lower disease free survival.ConclusionsWe provided evidence that a strong relationship occurs between α6β4 and ErbB-3 positivity in ERβ1-negative breast cancers. We also found that the association between ErbB-3 and P-Akt positivity mainly occurs in ERβ1-negative breast cancer derived from patients with lower DFS indicating that both receptors are clinically relevant in predicting the response to Tamoxifen.

NK cells expressing TIM-3 show a marked increase in IFNγ production in response to acute myeloid leukemia (AML) blast cells that endogenously express Gal-9. Herein, we demonstrate that NK cell-mediated production of IFNγ, induced by TIM-3/Gal-9 interaction and released in bone marrow microenvironment, is responsible for IDO1 expression in AML blasts. IDO1-expressing AML blasts consequently down-regulate NK cell degranulation activity, by sustaining leukemia immune escape. Furthermore, the blocking of TIM-3/Gal-9 interaction strongly down-regulates IFNγ-dependent IDO1 activity. Thus, the inhibition of TIM-3/Gal-9 immune check point, which affects NK cell-dependent IFNγ production and the consequent IDO1 activation, could usefully integrate current chemotherapeutic approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0134-4) contains supplementary material, which is available to authorized users.

Increased expression of A 6 B 4 integrin in several epithelial cancers promotes tumor progression; however, the mechanism underlying its transcriptional regulation remains unclear. Here, we show that depletion of homeodomaininteracting protein kinase 2 (HIPK2) activates B 4 transcription that results in a strong increase of B 4 -dependent mitogenactivated protein kinase and Akt phosphorylation, anchorageindependent growth, and invasion. In contrast, stabilization of HIPK2 represses B 4 expression in wild-type p53 (wtp53)-expressing cells but not in p53-null cells or cells expressing mutant p53, indicating that HIPK2 requires a wtp53 to inhibit B 4 transcription. Consistent with our in vitro findings, a strong correlation between B 4 overexpression and HIPK2 inactivation by cytoplasmic relocalization was observed in wtp53-expressing human breast carcinomas. Under loss of function of HIPK2 or p53, the p53 family members TAp63 and TAp73 strongly activate B 4 transcription. These data, by revealing that B 4 expression is transcriptionally repressed in tumors by HIPK2 and p53 to impair B 4 -dependent tumor progression, suggest that loss of p53 function favors the formation of coactivator complex with the TA members of the p53 family to allow B 4 transcription.