TAp73 represses NF-κB-mediated recruitment of tumor-associated macrophages in breast cancer.

Infiltration of tumor-promoting immune cells is a strong driver of tumor progression. Especially the accumulation of macrophages in the tumor microenvironment is known to facilitate tumor growth and to correlate with poor prognosis in many tumor types. TAp73, a member of the p53/p63/p73 family, acts as a tumor suppressor and has been shown to suppress tumor angiogenesis. However, what role TAp73 has in regulating immune cell infiltration is unknown. Here, we report that low levels of TAp73 correlate with an increased NF-κB-regulated inflammatory signature in breast cancer. Furthermore, we show that loss of TAp73 results in NF-κB hyperactivation and secretion of Ccl2, a known NF-κB target and chemoattractant for monocytes and macrophages. Importantly, TAp73-deficient tumors display an increased accumulation of protumoral macrophages that express the mannose receptor (CD206) and scavenger receptor A (CD204) compared to controls. The relevance of TAp73 expression in human breast carcinoma was further accentuated by revealing that TAp73 expression correlates negatively with the accumulation of protumoral CD163+ macrophages in breast cancer patient samples. Taken together, our findings suggest that TAp73 regulates macrophage accumulation and phenotype in breast cancer through inhibition of the NF-κB pathway.

ΔNp73 enhances HIF-1α protein stability through repression of the ECV complex.

Cellular responses to low oxygen conditions are mainly regulated by the Hypoxia-inducible factors (HIFs). Induction of HIF-1α in tumor cells activates the angiogenic switch and allows for metabolic adaptations. HIF-1α protein levels are tightly regulated through ubiquitin-mediated proteosomal degradation; however, high levels of HIF-1α is a common feature in many solid tumors and is thought to enhance cancer cell proliferation, migration, and survival. Here, we report that the oncogenic p73 isoform, ∆Np73, increases HIF-1α protein stability. We found that ∆Np73 represses expression of genes encoding subunits of the ECV complex, in particular Elongin C, Elongin B, Cullin 2, and Rbx1. The ECV complex is an E3 ligase complex responsible for polyubiquitinating HIF-1α. Loss of ∆Np73 increases ubiquitination of HIF-1α, leading to its degradation via the proteosomal pathway, and subsequent decrease of HIF-1α target genes. Taken together, our data demonstrates that high levels of ∆Np73 stabilize HIF-1α protein, allowing for it to accumulate and further potentiating its transcriptional activity and supporting tumor progression.

ΔNp73 regulates the expression of the multidrug-resistance genes ABCB1 and ABCB5 in breast cancer and melanoma cells - a short report.

PURPOSE: Multidrug resistance (MDR) is a major cause of treatment failure. In cancer cells, MDR is often caused by an increased efflux of therapeutic drugs mediated by an up-regulation of ATP binding cassette (ABC) transporters. It has previously been shown that oncogenic ΔNp73 plays an important role in chemo-resistance. Here we aimed at unraveling the role of ΔNp73 in regulating multidrug resistance in breast cancer and melanoma cells. METHODS: KEGG pathway analysis was used to identify pathways enriched in breast cancer samples with a high ΔNp73 expression. We found that the ABC transporter pathway was most enriched. The expression of selected ABC transporters was analyzed using qRT-PCR upon siRNA/shRNA-mediated knockdown or exogenous overexpression of ΔNp73 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, as well as in primary melanoma samples and in the melanoma-derived cell line SK-MEL-28. The ability to efflux doxorubicin and the concomitant effects on cell proliferation were assessed using flow cytometry and WST-1 assays. RESULTS: We found that high ΔNp73 levels correlate with a general up-regulation of ABC transporters in breast cancer samples. In addition, we found that exogenous expression of ΔNp73 led to an increase in the expression of ABCB1 and ABCB5 in the breast cancer-derived cell lines tested, while knocking down of ΔNp73 resulted in a reduction in ABCB1 and ABCB5 expression. In addition, we found that ΔNp73 reduction leads to an intracellular retention of doxorubicin in MDA-MB-231 and MCF7 cells and a concomitant decrease in cell proliferation. The effect of ΔNp73 on ABCB5 expression was further confirmed in metastases from melanoma patients and in the melanoma-derived cell line SK-MEL-28. CONCLUSIONS: Our data support a role for ΔNp73 in the multidrug-resistance of breast cancer and melanoma cells.

TAp73 suppresses tumor angiogenesis through repression of proangiogenic cytokines and HIF-1α activity.

The p53-family member TAp73 is known to function as a tumor suppressor and regulates genomic integrity, cellular proliferation, and apoptosis; however, its role in tumor angiogenesis is poorly understood. Here we demonstrate that TAp73 regulates tumor angiogenesis through repression of proangiogenic and proinflammatory cytokines. Importantly, loss of TAp73 results in highly vascularized tumors, as well as an increase in vessel permeability resulting from disruption of vascular endothelial-cadherin junctions between endothelial cells. In contrast, loss of the oncogenic p73 isoform ΔNp73 leads to reduced blood vessel formation in tumors. Furthermore, we show that up-regulated ΔNp73 levels are associated with increased angiogenesis in human breast cancer and that inhibition of TAp73 results in an accumulation of HIF-1α and up-regulation of HIF-1α target genes. Taken together, our data demonstrate that loss of TAp73 or ΔNp73 up-regulation activates the angiogenic switch that stimulates tumor growth and progression.

Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in the DNA damage response pathway.

Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and DeltaNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the DeltaNp73 isoform. Mice lacking DeltaNp73 (DeltaNp73(-/-) mice) are viable and fertile but display signs of neurodegeneration. Cells from DeltaNp73(-/-) mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in DeltaNp73(-/-) cells, we discovered a completely new role for DeltaNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that DeltaNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of DeltaNp73 expression show enhanced resistance to chemotherapy.

Regulation of tumor suppressor p53 at the RNA level.

p53 is a key tumor suppressor that triggers cell cycle arrest, senescence, or apoptosis in response to cellular stress. Frequent p53 mutation in human tumors allows survival, sustained growth, and tumor progression. p53 is expressed at low levels under normal conditions, due to rapid protein turnover. Stress signaling induces p53 protein stabilization through phosphorylation and other post-translational modifications. However, recent studies have demonstrated critical regulation of p53 at the mRNA level, mediated via both the 5'UTR and the 3'UTR and affecting both the stability and the translation efficiency of the p53 mRNA. Both proteins and microRNAs have been implicated in such regulation. The p53 target gene Wig-1 encodes a zinc finger protein that binds to double-stranded RNA and enhances p53 mRNA stability by binding to the 3'UTR in a positive feedback loop. Here, we shall summarize current knowledge about regulation of the p53 mRNA and discuss possible implications for cancer therapy.

The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element.

The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.

RIP2 contributes to Nod signaling but is not essential for T cell proliferation, T helper differentiation or TLR responses.

Receptor-interacting protein 2 (RIP2), also known as CARDIAK and RICK, has been reported to play a role in both adaptive T cell responses and innate immunity as a mediator in TLR signaling and nucleotide-binding oligomerization domain (Nod) signaling. Because initial reports remain controversial, we have further examined both innate and adaptive immune responses in RIP2-deficient mice on the C57BL/6 background. Despite the up-regulation of RIP2 after T cell activation, we could not detect any defect in T cell proliferation or Th1/Th2 responses in RIP2-KO mice. Furthermore, we found that TLR responses in RIP2-deficient macrophages were normal. However, our analysis showed that Nod signaling was impaired in macrophages from RIP2-deficient mice. In conclusion, our data demonstrate a critical role for RIP2 in Nod signaling, while T cell proliferation, T helper differentiation and TLR responses were unaffected by the absence of RIP2.

Identification of functional p53-binding motifs in the mouse wig-1 promoter.

We previously identified wig-1 as a p53-induced mouse gene that encodes a nuclear zinc finger protein with unknown function. To investigate whether wig-1 is a direct target of p53-dependent transactivation, a DNA fragment corresponding to the promoter region was cloned and sequenced. Three regions containing consensus p53-binding sites were identified. Two p53-binding motifs formed DNA-protein complexes with p53 and were able to drive p53-dependent transcription in a luciferase reporter assay. Our results demonstrate that wig-1 is a direct target of p53-mediated transcriptional transactivation.

The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity.

The p53-induced mouse wig-1 gene encodes a Cys2His2-type zinc finger protein of unknown function. The zinc fingers in wig-1 are connected by long (56-75) amino acid linkers. This distribution of zinc finger domains resembles that of the previously described double-stranded (ds)RNA-binding proteins dsRBP-ZFa and JAZ. Ectopically expressed FLAG-tagged mouse wig-1 protein localized to nuclei and in some cells to nucleoli, whereas GFP-tagged mouse wig-1 localized primarily to nucleoli. Electrophoretic mobility shift assay using a recombinant GST-wig-1 fusion protein showed that wig-1 preferentially binds dsRNA rather than single-stranded RNA or dsDNA. A set of deletion/truncation mutants of wig-1 was tested to determine the dsRNA-binding domain(s) or region(s) in wig-1 that is involved in the stabilization of wig-1-dsRNA complexes in vitro. This revealed that the first zinc finger in wig-1 is essential for binding to dsRNA, whereas zinc fingers 2 and 3 are dispensable. wig-1 protein expressed in mammalian cells also showed a high affinity for dsRNA. wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA binding plays a role in the p53-dependent stress response.