Andrea Lunardi

p53-family proteins and their regulators: hubs and spokes in tumor suppression.

The tumor suppressor p53 is a central hub in a molecular network controlling cell proliferation and death in response to potentially oncogenic conditions, and a wide array of covalent modifications and protein interactions modulate the nuclear and cytoplasmic activities of p53. The p53 relatives, p73 and p63, are entangled in the same regulatory network, being subject at least in part to the same modifications and interactions that convey signals on p53, and actively contributing to the resulting cellular output. The emerging picture is that of an interconnected pathway, in which all p53-family proteins are involved in the response to oncogenic stress and physiological inputs. Therefore, common and specific interactors of p53-family proteins can have a wide effect on function and dysfunction of this pathway. Many years of research have uncovered an impressive number of p53-interacting proteins, but much less is known about protein interactions of p63 and p73. Yet, many interactors may be shared by multiple p53-family proteins, with similar or different effects. In this study we review shared interactors of p53-family proteins with the aim to encourage research into this field; this knowledge promises to unveil regulatory elements that could be targeted by a new generation of molecules, and allow more efficient use of currently available drugs for cancer treatment.

Cancer-Associated PTEN Mutants Act in a Dominant-Negative Manner to Suppress PTEN Protein Function

PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here, we show that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on PtdIns(3,4,5)P3. We demonstrate that catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, we generated Pten knockin mice harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous Pten(C124S/+) and Pten(G129E/+) cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten(+/-) counterparts, whereas this difference is no longer apparent between Pten(C124S/-) and Pten(-/-) cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. Our findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN.

A co-clinical approach identifies mechanisms and potential therapies for androgen deprivation resistance in prostate cancer

Here we report an integrated analysis that leverages data from treatment of genetic mouse models of prostate cancer along with clinical data from patients to elucidate new mechanisms of castration resistance. We show that castration counteracts tumor progression in a Pten loss–driven mouse model of prostate cancer through the induction of apoptosis and proliferation block. Conversely, this response is bypassed with deletion of either Trp53 or Zbtb7a together with Pten, leading to the development of castration-resistant prostate cancer (CRPC). Mechanistically, the integrated acquisition of data from mouse models and patients identifies the expression patterns of XAF1, XIAP and SRD5A1 as a predictive and actionable signature for CRPC. Notably, we show that combined inhibition of XIAP, SRD5A1 and AR pathways overcomes castration resistance. Thus, our co-clinical approach facilitates the stratification of patients and the development of tailored and innovative therapeutic treatments.

Zbtb7a suppresses prostate cancer through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion

Zbtb7a has previously been described as a powerful proto-oncogene. Here we unexpectedly demonstrate that Zbtb7a has a critical oncosuppressive role in the prostate. Prostate-specific inactivation of Zbtb7a leads to a marked acceleration of Pten loss–driven prostate tumorigenesis through bypass of Pten loss–induced cellular senescence (PICS). We show that ZBTB7A physically interacts with SOX9 and functionally antagonizes its transcriptional activity on key target genes such as MIA, which is involved in tumor cell invasion, and H19, a long noncoding RNA precursor for an RB-targeting microRNA. Inactivation of Zbtb7a in vivo leads to Rb downregulation, PICS bypass and invasive prostate cancer. Notably, we found that ZBTB7A is genetically lost, as well as downregulated at both the mRNA and protein levels, in a subset of human advanced prostate cancers. Thus, we identify ZBTB7A as a context-dependent cancer gene that can act as an oncogene in some contexts but also has oncosuppressive-like activity in PTEN-null tumors.

In Vivo Role of INPP4B in Tumor and Metastasis Suppression through Regulation of PI3K–AKT Signaling at Endosomes

UNLABELLED The phosphatases PTEN and INPP4B have been proposed to act as tumor suppressors by antagonizing PI3K-AKT signaling and are frequently dysregulated in human cancer. Although PTEN has been extensively studied, little is known about the underlying mechanisms by which INPP4B exerts its tumor-suppressive function and its role in tumorigenesis in vivo. Here, we show that a partial or complete loss of Inpp4b morphs benign thyroid adenoma lesions in Pten heterozygous mice into lethal and metastatic follicular-like thyroid cancer (FTC). Importantly, analyses of human thyroid cancer cell lines and specimens reveal INPP4B downregulation in FTC. Mechanistically, we find that INPP4B, but not PTEN, is enriched in the early endosomes of thyroid cancer cells, where it selectively inhibits AKT2 activation and in turn tumor proliferation and anchorage-independent growth. We therefore identify INPP4B as a novel tumor suppressor in FTC oncogenesis and metastasis through localized regulation of the PI3K-AKT pathway at the endosomes. SIGNIFICANCE Although both PTEN and INPP4B can inhibit PI3K-AKT signaling through their lipid phosphatase activities, here we demonstrate lack of an epistatic relationship between the two tumor suppressors. Instead, the qualitative regulation of PI3K-AKT2 signaling by INPP4B provides a mechanism for their cooperation in suppressing thyroid tumorigenesis and metastasis.

Vulnerabilities of PTEN–TP53-Deficient Prostate Cancers to Compound PARP–PI3K Inhibition

UNLABELLED Prostate cancer is the most prevalent cancer in males, and treatment options are limited for advanced forms of the disease. Loss of the PTEN and TP53 tumor suppressor genes is commonly observed in prostate cancer, whereas their compound loss is often observed in advanced prostate cancer. Here, we show that PARP inhibition triggers a p53-dependent cellular senescence in a PTEN-deficient setting in the prostate. Surprisingly, we also find that PARP-induced cellular senescence is morphed into an apoptotic response upon compound loss of PTEN and p53. We further show that superactivation of the prosurvival PI3K-AKT signaling pathway limits the efficacy of a PARP single-agent treatment, and that PARP and PI3K inhibitors effectively synergize to suppress tumorigenesis in human prostate cancer cell lines and in a Pten/Trp53-deficient mouse model of advanced prostate cancer. Our findings, therefore, identify a combinatorial treatment with PARP and PI3K inhibitors as an effective option for PTEN-deficient prostate cancer. SIGNIFICANCE The paucity of therapeutic options in advanced prostate cancer displays an urgent need for the preclinical assessment of novel therapeutic strategies. We identified differential therapeutic vulnerabilities that emerge upon the loss of both PTEN and p53, and observed that combined inhibition of PARP and PI3K provides increased efficacy in hormone-insensitive advanced prostate cancer.

A genome-scale protein interaction profile of Drosophila p53 uncovers additional nodes of the human p53 network

The genome of the fruitfly Drosophila melanogaster contains a single p53-like protein, phylogenetically related to the ancestor of the mammalian p53 family of tumor suppressors. We reasoned that a comprehensive map of the protein interaction profile of Drosophila p53 (Dmp53) might help identify conserved interactions of the entire p53 family in man. Using a genome-scale in vitro expression cloning approach, we identified 91 previously unreported Dmp53 interactors, considerably expanding the current Drosophila p53 interactome. Looking for evolutionary conservation of these interactions, we tested 41 mammalian orthologs and found that 37 bound to one or more p53-family members when overexpressed in human cells. An RNAi-based functional assay for modulation of the p53 pathway returned five positive hits, validating the biological relevance of these interactions. One p53 interactor is GTPBP4, a nucleolar protein involved in 60S ribosome biogenesis. We demonstrate that GTPBP4 knockdown induces p53 accumulation and activation in the absence of nucleolar disruption. In breast tumors with wild-type p53, increased expression of GTPBP4 correlates with reduced patient survival, emphasizing a potential relevance of this regulatory axis in cancer.

Modification of Drosophila p53 by SUMO modulates its transactivation and pro-apoptotic functions

Conjugation to SUMO is a reversible post-translational modification that regulates several transcription factors involved in cell proliferation, differentiation, and disease. The p53 tumor suppressor can be modified by SUMO-1 in mammalian cells, but the functional consequences of this modification are unclear. Here, we demonstrate that the Drosophila homolog of human p53 can be efficiently sumoylated in insect cells. We identify two lysine residues involved in SUMO attachment, one at the C terminus, between the DNA binding and oligomerization domains, and one at the N terminus of the protein. We find that sumoylation helps recruit Drosophila p53 to nuclear dot-like structures that can be marked by human PML and the Drosophila homologue of Daxx. We demonstrate that mutation of both sumoylation sites dramatically reduces the transcriptional activity of p53 and its ability to induce apoptosis in transgenic flies, providing in vivo evidence that sumoylation is critical for Drosophila p53 function.

Role of LRF/Pokemon in lineage fate decisions

In the human genome, 43 different genes are found that encode proteins belonging to the family of the POK (poxvirus and zinc finger and Krüppel)/ZBTB (zinc finger and broad complex, tramtrack, and bric à brac) factors. Generally considered transcriptional repressors, several of these genes play fundamental roles in cell lineage fate decision in various tissues, programming specific tasks throughout the life of the organism. Here, we focus on functions of leukemia/lymphoma-related factor/POK erythroid myeloid ontogenic factor, which is probably one of the most exciting and yet enigmatic members of the POK/ZBTB family.

A role for PML in innate immunity

The promyelocytic leukemia gene (PML) of acute promyelocytic leukemia is an established tumor suppressor gene with critical functions in growth suppression, induction of apoptosis, and cellular senescence. Interestingly, although less studied, PML seems to play a key role also in immune response to viral infection. Herein, we report that Pml(-/-) mice spontaneously develop an atypical invasive and lethal granulomatous lesion known as botryomycosis (BTM). In Pml(-/-) mice, BTM is the result of impaired function of macrophages, whereby they fail to become activated and are thus unable to clear pathogenic microorganisms. Accordingly, Pml(-/-) mice are resistant to lipopolysaccharide (LPS)-induced septic shock as a result of an ineffective production of cytokines and chemokines, suggesting a role for PML in the innate immune Toll-like receptor (TLR)/NF-κB prosurvival pathway. These results not only shed light on a new fundamental function of PML in innate immunity, but they also point to a proto-oncogenic role for PML in certain cellular and pathological contexts.