Martin Monte

The death substrate Gas2 binds m‐calpain and increases susceptibility to p53‐dependent apoptosis

Gas2 is a caspase‐3 substrate that plays a role in regulating microfilament and cell shape changes during apoptosis. Here we provide evidence that overexpression of Gas2 efficiently increases cell susceptibility to apoptosis following UV irradiation, etoposide and methyl methanesulfonate treatments, and that these effects are dependent on increased p53 stability and transcription activity. To investigate possible pathways linking Gas2 to p53, a yeast two‐hybrid screen swas performed, indicating m‐calpain as a strong Gas2‐ interacting protein. Moreover, we demonstrate that Gas2 physically interacts with m‐calpain in vivo and that recombinant Gas2 inhibits calpain‐dependent processing of p53. Importantly, the Gas2 dominant‐negative form (Gas2Δ171–314) that binds calpain but is unable to inhibit its activity abrogates Gas2s ability to stabilize p53, to enhance p53 transcriptional activity and to induce p53‐dependent apoptosis. Finally, we show that Gas2 is able to regulate the levels of p53 independently of Mdm2 status, suggesting that, like calpastatin, it may enhance p53 stability by inhibiting calpain activity.

wt p53 dependent expression of a membrane-associated isoform of adenylate kinase.

Six novel p53-inducible transcripts were recently cloned from Val5, a murine cell line stably expressing a temperature-sensitive p53 allele. One of the isolated clones represented a novel isoform of cytosolic adenylate kinase (AK1), a highly conserved monomeric enzyme involved in cellular homeostasis of adenine nucleotides. The corresponding protein, which we named AK1β, was specifically induced upon activation of wt p53 in Val5 cells. The AK1β protein differs from cytoplasmic AK1 by having 18 extra amino acids at the N-terminus. The extra residues in AK1β provide a consensus signal for N-terminal myristoylation; as expected, AK1β was shown to localize to the plasma membrane. The human AK1 gene contains several consensus p53 binding sites and we report that p53-dependent induction of the alternative AK1β transcript also occurs in human cells. By using antisense ablation experiments in Val5 fibroblasts we show that AK1β plays a relevant role in the establishment of reversible cell-cycle arrest as induced by p53 in these cells. These findings suggest that within a p53-dependent genetic program, a specific isoform of adenylate kinase has a previously undescribed growth-regulatory function, which might not necessarily require its best characterized biochemical activity.

GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

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.

Human GTSE-1 Regulates p21 CIP1/WAF1 Stability Conferring Resistance to Paclitaxel Treatment *□

p21CIP1/WAF1 belongs to the CIP/KIP family of Cdk inhibitors, and its expression is tightly controlled during the cell cycle, mainly by transcriptional and post-translational mechanisms. Fine regulation of p21CIP1/WAF1 levels is critical for cell cycle control and for cellular response to stress. In the present work, we describe a novel mechanism to modulate p21CIP1/WAF1 levels mediated by the human GTSE-1 (G2 and S phase-expressed-1) protein. Our results provide evidence that hGTSE-1 protects p21CIP1/WAF1 from proteasome-dependent degradation as part of a functional complex containing the Hsp90-binding TPR protein WISp39. We further show that the hGTSE-1 N-terminal portion is sufficient for p21CIP1/WAF1 binding and stabilization. Finally, we demonstrate that hGTSE-1 mediated-p21CIP1/WAF1 stabilization is clearly involved in the ability of cells to counteract cytotoxicity induced by the microtubule poison paclitaxel.

Cell-cycle regulation of the p53-inducible gene B99

B99 is a p53‐inducible gene whose accumulation upon p53 activation is restricted to late S/G2 cells. Here we have analyzed B99 regulation during the cell cycle in murine cells with or without functional p53. We report that B99 accumulates in late S/G2 phase, is phosphorylated in mitosis, and disappears in G1 phase, regardless of the status of p53. As a complement to this observation, we show that B99 is not induced by p53 in quiescent cells. Therefore, B99 expression is modulated both by cell‐cycle regulatory mechanisms and by p53, and p53 can increase the cellular levels of B99 only during the window of the cell cycle when it is normally expressed. On the basis of these observations we rename B99 Gtse‐1 (G‐two‐ and S‐phase‐expressed).

Cloning, chromosome mapping and functional characterization of a human homologue of murine Gtse-1 (B99) gene

Murine Gtse-1 (G(2) and S phase expressed protein), previously named B99, is a wt-p53 inducible gene that encodes a microtubule-localized protein which is able to induce G(2)/M phase accumulation when ectopically expressed. Here we report the cloning and characterization of a new cDNA (GTSE-1) encoding a human homologue of the mouse Gtse-1 protein. Chromosome mapping of mouse and human genes assigned Gtse-1 to chromosome 15 and GTSE-1 to chromosome 22q13.2-q13.3 in a region with conserved synteny to that where Gtse-1 mapped. Analysis of the genomic structure revealed that GTSE-1 contains at least 11 exons and 10 introns, spanning approximately 33kb of genomic DNA. Similar to murine Gtse-1, the product of GTSE-1 localized to the microtubules, was able to delay G(2)/M progression when ectopically expressed and was cell cycle regulated. Taken together, these results indicate GTSE-1 as the human functional homologue of murine Gtse-1.

Cloning and characterization of the C. elegans gas1 homolog: phas-1

Among the set of genes expressed during the quiescent G0 phase of the cell cycle (gas genes), gas1 encodes for a GPI anchor protein associated to the plasma membrane, which is able to induce growth arrest when overexpressed in proliferating fibroblasts. In this report we describe the isolation and characterization of a gas1 Caenorhabditis elegans homolog, phas-1, that seems to be transcribed as an operon together with a gene encoding for a protein similar to human acid ceramidases. Phas-1 structure is very similar to its mammalian homolog conserving almost all cysteine residues and it is expressed in the pharynx from its early formation, in the two-fold embryo, until the adult stage. Surprisingly, while phas-1 is expressed in all developmental stages, with the exception of the dauer larva, the ceramidase-like encoding gene, co-expressed in the same operon together with phas-1, is absent in embryos and is very abundantly expressed in the dauer larva. Overexpression of phas-1 in growing NIH3T3 fibroblasts is able to inhibit the S-phase entry in a similar manner as its murine homolog. On the other hand, when phas-1 is overexpressed or ablated in C. elegans, no specific phenotype due to its transcription alteration can be observed, despite its localized expression suggesting a role in pharynx function or development.

GTSE1: a novel TEAD4-E2F1 target gene involved in cell protrusions formation in triple-negative breast cancer cell models

GTSE1 over-expression has been reported as a potential marker for metastasis in various types of malignancies, including breast cancer. Despite this, the transcriptional regulation of this protein and the causes of its misregulation in tumors remain largely unknown. The aims of this work were to elucidate how GTSE1 is regulated at the transcriptional level and to clarify the mechanism underlying GTSE1-dependent cell functions in triple-negative breast cancer (TNBC).Here, we identified GTSE1 as a novel target gene of the TEAD4 transcription factor, highlighting a role for the YAP and TAZ coactivators in the transcriptional regulation of GTSE1.Moreover, we found that TEAD4 controls the formation of cell protrusions required for cell migration through GTSE1, unveiling a relevant effector role for this protein in the TEAD-dependent cellular functions and confirming TEAD4 role in promoting invasion and metastasis in breast cancer.Finally, we highlighted a role for the pRb-E2F1 pathway in the control of GTSE1 transcription and observed that treatment with drugs targeting the pRb-E2F1 or YAP/TAZ-TEAD pathways dramatically downregulated the expression levels of GTSE1 and of other genes involved in the formation of metastasis, suggesting their potential use in the treatment of TNBC.GTSE1 over-expression has been reported as a potential marker for metastasis in various types of malignancies, including breast cancer. Despite this, the transcriptional regulation of this protein and the causes of its misregulation in tumors remain largely unknown. The aims of this work were to elucidate how GTSE1 is regulated at the transcriptional level and to clarify the mechanism underlying GTSE1-dependent cell functions in triple-negative breast cancer (TNBC). Here, we identified GTSE1 as a novel target gene of the TEAD4 transcription factor, highlighting a role for the YAP and TAZ coactivators in the transcriptional regulation of GTSE1. Moreover, we found that TEAD4 controls the formation of cell protrusions required for cell migration through GTSE1, unveiling a relevant effector role for this protein in the TEAD-dependent cellular functions and confirming TEAD4 role in promoting invasion and metastasis in breast cancer. Finally, we highlighted a role for the pRb-E2F1 pathway in the control of GTSE1 transcription and observed that treatment with drugs targeting the pRb-E2F1 or YAP/TAZ-TEAD pathways dramatically downregulated the expression levels of GTSE1 and of other genes involved in the formation of metastasis, suggesting their potential use in the treatment of TNBC.