The MAGE gene family is characterized by a conserved domain (MAGE Homology Domain). A subset of highly homologous MAGE genes (group A; MAGE-A) belong to the chromosome X-clustered cancer͞testis antigens. MAGE-A genes are normally expressed in the human germ line and overexpressed in various tumor types; however, their biological function is largely unknown. Here we present evidence indicating that MageA2 protein, belonging to the MAGE-A subfamily, confers wild-type-p53-sensitive resistance to etoposide (ET) by inducing a novel p53 inhibitory loop involving recruitment of histone deacetylase 3 (HDAC3) to MageA2͞p53 complex, thus strongly down-regulating p53 transactivation function. In fact, enhanced MageA2 protein levels, in addition to ET resistance, correlate with impaired acetylation of both p53 and histones surrounding p53-binding sites. Association between MAGE-A expression levels and resistance to ET treatment is clearly shown in short-term cell lines obtained from melanoma biopsies harboring wild-type-p53, whereas cells naturally, or siRNAmediated expressing low MAGE-A levels, correlate with enhanced p53-dependent sensitivity to ET. In addition, combined trichostatin A͞ET treatment in melanoma cells expressing high MAGE-A levels reestablishes p53 response and reverts the chemoresistance.

The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

MAGE-A genes are a subfamily of the melanoma antigen genes (MAGEs), whose expression is restricted to tumor cells of different origin and normal tissues of the human germline. Although the specific function of individual MAGE-A proteins is being currently explored, compelling evidence suggest their involvement in the regulation of different pathways during tumor progression. We have previously reported that MageA2 binds histone deacetylase (HDAC)3 and represses p53-dependent apoptosis in response to chemotherapeutic drugs. The promyelocytic leukemia (PML) tumor suppressor is a regulator of p53 acetylation and function in cellular senescence. Here, we demonstrate that MageA2 interferes with p53 acetylation at PMLnuclear bodies (NBs) and with PMLIV-dependent activation of p53. Moreover, a fraction of MageA2 colocalizes with PML-NBs through direct association with PML, and decreases PMLIV sumoylation through an HDAC-dependent mechanism. This reduction in PML post-translational modification promotes defects in PML-NBs formation. Remarkably, we show that in human fibroblasts expressing RasV12 oncogene, MageA2 expression decreases cellular senescence and increases proliferation. These results correlate with a reduction in NBs number and an impaired p53 response. All these data suggest that MageA2, in addition to its anti-apoptotic effect, could have a novel role in the early progression to malignancy by interfering with PML/p53 function, thereby blocking the senescence program, a critical barrier against cell transformation.

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cCalpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy, through the modulating cleavage of specific substrates. Ubiquitous microcalpain (-calpain) and millicalpain (m-calpain) are heterodimers composed of catalytic subunits encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Here we show that calpain is required for the stability of the deubiquitinating enzyme USP1 in several cell lines. USP1 modulates DNA replication polymerase choice and repair by deubiquitinating PCNA. The ubiquitinated form of the USP1 substrate PCNA is stabilized in CAPNS1-depleted U2OS cells and mouse embryonic fibroblasts (MEFs), favoring polymeraseloading on chromatin and increased mutagenesis. USP1 degradation directed by the cell cycle regulator APC/C cdh1 , which marks USP1 for destruction in the G 1 phase, is upregulated in CAPNS1-depleted cells. USP1 stability can be rescued upon forced expression of calpain-activated Cdk5/p25, previously reported as a cdh1 repressor. These data suggest that calpain stabilizes USP1 by activating Cdk5, which in turn inhibits cdh1 and, consequently, USP1 degradation. Altogether these findings point to a connection between the calpain system and the ubiquitin pathway in the regulation of DNA damage response and place calpain at the interface between cell cycle modulation and DNA repair. Calpains regulate a wide spectrum of biological functions, including migration, adhesion, apoptosis, secretion, and autophagy through the modulated cleavage of specific substrates (reviewed in references 1-3). Ubiquitous microcalpain (-calpain) and millicalpain (m-calpain) are heterodimers composed of a catalytic subunit encoded, respectively, by CAPN1 and CAPN2 and a regulatory subunit encoded by CAPNS1. Both -calpain and mcalpain are negatively modulated by calpastatin. By a proteomic approach, we identified USP1 deubiquitinase as a CAPNS1-interacting protein. USP1 is a key modulator of DNA repair, partly through deubiquitination of its known targets FANCD2 (4, 5) and PCNA (6). Usp1 knockout (KO) mice have a severe phenotype and die soon after birth (7). Usp1 Ϫ/Ϫ cells are defective in FANCD2 focus formation and are hypersensitive to DNA damage (8). PCNA ubiquitination is higher in USP1-depleted cells than in control cells, thus leading to recruitment of error-prone, translesion DNA synthesis (TLS) polymerases and the consequent increase in mutation rate (6). USP1 promotes inhibitor of DNA binding (ID) protein stability and stem cell-like characteristics in osteosarcoma and is required for normal skeletogenesis (9). Interestingly, mice lacking CAPNS1 in cells of the osteoblast lineage are defective in bone development and remodeling in vivo (10).UV light irradiation activates hUSP1 autocleavage at glycines 670 and 671, inducing its subsequent proteasomal degradation (6). USP1 mutants with mutation in the catalytic cysteine 90 or in the autocleavage sites are more stable but can still be degraded in the cell (5), s...