Δ133p53ß isoform pro-invasive activity is regulated through an aggregation-dependent mechanism in cancer cells.

The p53 isoform, Δ133p53ß, is critical in promoting cancer. Here we report that Δ133p53ß activity is regulated through an aggregation-dependent mechanism. Δ133p53ß aggregates were observed in cancer cells and tumour biopsies. The Δ133p53ß aggregation depends on association with interacting partners including p63 family members or the CCT chaperone complex. Depletion of the CCT complex promotes accumulation of Δ133p53ß aggregates and loss of Δ133p53ß dependent cancer cell invasion. In contrast, association with p63 family members recruits Δ133p53ß from aggregates increasing its intracellular mobility. Our study reveals novel mechanisms of cancer progression for p53 isoforms which are regulated through sequestration in aggregates and recruitment upon association with specific partners like p63 isoforms or CCT chaperone complex, that critically influence cancer cell features like EMT, migration and invasion.

∆133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling.

∆122p53 mice (a model of ∆133p53 isoform) are tumour-prone, have extensive inflammation and elevated serum IL-6. To investigate the role of IL-6 we crossed ∆122p53 mice with IL-6 null mice. Here we show that loss of IL-6 reduced JAK-STAT signalling, tumour incidence and metastasis. We also show that ∆122p53 activates RhoA-ROCK signalling leading to tumour cell invasion, which is IL-6-dependent and can be reduced by inhibition of JAK-STAT and RhoA-ROCK pathways. Similarly, we show that Δ133p53 activates these pathways, resulting in invasive and migratory phenotypes in colorectal cancer cells. Gene expression analysis of colorectal tumours showed enrichment of GPCR signalling associated with ∆133TP53 mRNA. Patients with elevated ∆133TP53 mRNA levels had a shorter disease-free survival. Our results suggest that ∆133p53 promotes tumour invasion by activation of the JAK-STAT and RhoA-ROCK pathways, and that patients whose tumours have high ∆133TP53 may benefit from therapies targeting these pathways.

Correction: The p53 isoform delta133p53ß regulates cancer cell apoptosis in a RhoB-dependent manner.

[This corrects the article DOI: 10.1371/journal.pone.0172125.].

The p53 isoform delta133p53ß regulates cancer cell apoptosis in a RhoB-dependent manner.

The TP53 gene plays essential roles in cancer. Conventionally, wild type (WT) p53 is thought to prevent cancer development and metastasis formation, while mutant p53 has transforming abilities. However, clinical studies failed to establish p53 mutation status as an unequivocal predictive or prognostic factor of cancer progression. The recent discovery of p53 isoforms that can differentially regulate cell cycle arrest and apoptosis suggests that their expression, rather than p53 mutations, could be a more clinically relevant biomarker in patients with cancer. In this study, we show that the p53 isoform delta133p53ß is involved in regulating the apoptotic response in colorectal cancer cell lines. We first demonstrate delta133p53ß association with the small GTPase RhoB, a well-described anti-apoptotic protein. We then show that, by inhibiting RhoB activity, delta133p53ß protects cells from camptothecin-induced apoptosis. Moreover, we found that high delta133p53 mRNA expression levels are correlated with higher risk of recurrence in a series of patients with locally advanced rectal cancer (n = 36). Our findings describe how a WT TP53 isoform can act as an oncogene and add a new layer to the already complex p53 signaling network.

TP53 drives invasion through expression of its Δ133p53ß variant.

TP53 is conventionally thought to prevent cancer formation and progression to metastasis, while mutant TP53 has transforming activities. However, in the clinic, TP53 mutation status does not accurately predict cancer progression. Here we report, based on clinical analysis corroborated with experimental data, that the p53 isoform Δ133p53ß promotes cancer cell invasion, regardless of TP53 mutation status. Δ133p53ß increases risk of cancer recurrence and death in breast cancer patients. Furthermore Δ133p53ß is critical to define invasiveness in a panel of breast and colon cell lines, expressing WT or mutant TP53. Endogenous mutant Δ133p53ß depletion prevents invasiveness without affecting mutant full-length p53 protein expression. Mechanistically WT and mutant Δ133p53ß induces EMT. Our findings provide explanations to 2 long-lasting and important clinical conundrums: how WT TP53 can promote cancer cell invasion and reciprocally why mutant TP53 gene does not systematically induce cancer progression.

The p53 isoform Δ133p53ß promotes cancer stem cell potential.

Cancer stem cells (CSC) are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53ß, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53ß stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53ß expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53ß in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53ß supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53ß isoform.

Cyclin A2, Rho GTPases and EMT.

Cell cycle regulators, such as cyclins, are often upregulated in many proliferative disorders, and Cyclin A2 is generally considered as a marker of aggressive cancers. Our recent work, which revealed decreased expression of Cyclin A2 upon metastasis of colorectal cancer, suggests a more complicated situation. Consistent with this, we identified a role for Cyclin A2, via RhoA, in regulation of the actin cytoskeleton and the control of cell invasion. Cyclin A2 also regulates spindle orientation which, when misoriented, could disrupt cell polarity and favor cancer cell detachment from the tumor as part of a transforming process, such as epithelial to mesenchymal transition (EMT). During EMT, cells undergo morphological and molecular changes toward a mesenchymal phenotype. Upregulation, or increased activity of some Rho GTPases, such as Cdc42, Rac1 or RhoC, increases the invasive potential of these cells. This correlates with the inverse relationship between RhoA and RhoC activities we observed in an epithelial cell type. Altogether, these observations raise the possibility that Cyclin A2 is instrumental in preventing EMT and therefore cancers of epithelial tissues.

A novel function for Cyclin A2: control of cell invasion via RhoA signaling.

Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration.

Cyclin A2: a genuine cell cycle regulator?

Abstract Cyclin A2 belongs to the core cell cycle regulators and participates in the control of both S phase and mitosis. However, several observations suggest that it is also endowed with other functions, and our recent data shed light on its involvement in cytoskeleton dynamic and cell motility. From the transcription of its gene to its posttranslational modifications, cyclin A2 regulation reveals the complexity of the regulatory network shaping cell cycle progression. We summarize our current knowledge on this cell cycle regulator and discuss recent findings raising the possibility that cyclin A2 might play a much broader role in epithelial tissues homeostasis.

Cyclin A2 mutagenesis analysis: a new insight into CDK activation and cellular localization requirements.

Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved.

Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice.

Experimental and clinical evidence indicate that bone marrow cells participate in the process of new blood vessel formation. However, the molecular mechanisms underlying their recruitment and their exact role are still elusive. Here, we show that bone marrow cells are recruited to the sites of neoangiogenesis through the neuropilin-1 (NP-1) receptor and that they are essential for the maturation of the activated endothelium and the formation of arteries in mice. By exploiting adeno-associated virus vector-mediated, long-term in vivo gene expression, we show that the 165-aa isoform of VEGF, which both activates the endothelium and recruits NP-1+ myeloid cells, is a powerful arteriogenic agent. In contrast, neither the shortest VEGF121 isoform, which does not bind NP-1 and thus does not recruit bone marrow cells, nor semaphorin 3A, which attracts cells but inhibits endothelial activation, are capable of sustaining arterial formation. Bone marrow myeloid cells are not arteriogenic per se nor are they directly incorporated in the newly formed vasculature, but they contribute to arterial formation through a paracrine effect ensuing in the activation and proliferation of tissue-resident smooth muscle cells.

Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages.

Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

A novel myogenic cell line with phenotypic properties of muscle progenitors.

Skeletal myogenesis is a multistep process starting with progenitor cell proliferation, followed by their exit from the cell cycle, differentiation, alignment, and fusion to form multinucleated myotubes, typical of the differentiated muscle tissue. While the molecular players involved in early myogenesis have been extensively characterized, information about the later steps of the process is scanty. Here, we describe a novel myogenic cell line (MYOP7), composed of highly proliferating Sca-1+ muscle precursor cells, which can be induced to terminally differentiate into spontaneously contracting multinucleated myotubes. By performing high-density microarray analysis on these cells, we identified a series of genes, differentially expressed in proliferating vs differentiating conditions, which are candidates to play a major role in the later phase of myogenesis. In addition, we confirmed that the late stages of muscle differentiation are characterized by a marked upregulation of the cellular receptors for the vascular endothelial growth factor.

In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature.

Although the angiogenic effect of vascular endothelial growth factor (VEGF) is widely recognized, a central question concerns whether the vessels formed on its overexpression effectively increase tissue perfusion in vivo. To explore this issue, here we exploit AAV vectors to obtain the prolonged expression of VEGF and angiopoietin-1 (Ang1) in rat skeletal muscle. Over a period of 6 months, muscle blood flow (MBF) and vascular permeability were measured by positron emission tomography and single-photon emission computed tomography, respectively. All measurements were performed under resting conditions and after electrically induced muscle exercise. Despite the potent angiogenic effect of VEGF, documented by vessel counting and intravascular volume assessment, the expression of this factor did not improve resting MBF, and it even decreased perfusion after exercise. This deleterious effect was related to the formation of leaky vascular lacunae, which accounted for the occurrence of arteriovenous shunts that excluded the downstream microcirculation. These effects were significantly counteracted by the coinjection of VEGF and Ang1, which determined a marked increase in resting MBF and, most notably, a significant improvement after exercise that persisted over time. Taken together, these results challenge the effectiveness of VEGF as a sole factor to induce angiogenesis and suggest the use of factor combinations to achieve competent vessel formation.

Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis.

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.

Adeno-associated virus-mediated transduction of VEGF165 improves cardiac tissue viability and functional recovery after permanent coronary occlusion in conscious dogs.

We have previously shown that VEGF165 gene delivery into ischemic skeletal muscle exerts not only proangiogenic, but also remarkable antiapoptotic and proregenerative activity. The aim of this study was to determine whether recombinant adeno-associated virus (rAAV)-mediated gene delivery of VEGF165 into cardiac muscle, during acute myocardial infarction, exerts a protective effect to promote long-term functional recovery. Acute infarction of the anterior LV wall was induced in 12 chronically instrumented dogs by permanent occlusion of the LAD coronary artery. Four hours after occlusion, rAAV-VEGF165 or rAAV-LacZ (n=6 each; 5x10(12) viral particles per animal) was directly injected with an echo-guided needle into the dysfunctional cardiac wall. LV and arterial pressure, dP/dtmax, and ejection fraction were not significantly different between the two groups over time. In contrast, in the infarcted region, at four weeks after infarction, fractional shortening was 75+/-18% and -3+/-15% of baseline and length-pressure area was 54+/-15% and 0.8+/-15% of baseline in VEGF165 versus LacZ, respectively (P<0.05). Histological analysis of the border regions showed a marked increase in the number of alpha-SMA-positive arterioles (68+/-2.8 versus 100+/-3.8 vessels per microscopic field in LacZ and VEGF165 group, respectively; P<0.05). In both groups, the receptor VEGFR-2 was diffusely expressed on the surviving cardiomyocytes and, consistently, myocardial viability was significantly improved in the VEGF165-treated group, with several troponin T-expressing cardiomyocytes displaying nuclear positivity for the proliferation marker PCNA. Altogether, our results indicate that VEGF165 gene delivery exerts a marked beneficial action by enhancing both arteriologenesis and cardiomyocyte viability in infarcted myocardium.

Bone marrow mononuclear cells are recruited to the sites of VEGF-induced neovascularization but are not incorporated into the newly formed vessels.

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel formation during both vasculogenesis and angiogenesis. The prolonged expression of VEGF in the normoperfused skeletal muscles of adult rodents after gene transfer using AAV vectors induces the formation of a large set of new capillaries and small arteries. Notably, this process is accompanied by the massive infiltration by mononuclear cells. This observation raises the possibility that these cells might represent circulating progenitors that are eventually incorporated in the newly formed vessels. Here we explore this possibility by exploiting 4 different experimental models based on (a) the transplantation of male bone marrow into female recipients; (b) the transplantation of Tie2-GFP transgenic bone marrow; (c) the transplantation of bone marrow in the presence of erythropoietin (EPO), a mobilizer of endothelial progenitor cells (EPCs); and (d) the reimplantation of ex vivo-expanded EPCs. In all 4 models, VEGF acted as a powerful attractor of bone marrow-derived mononuclear cells, bearing different myeloid and endothelial markers proper of the EPCs to the sites of neovascularization. In no case, however, were the attracted cells incorporated in the newly formed vasculature. These observations indicate that new blood vessel formation induced by VEGF occurs through bona fide sprouting angiogenesis; the contribution of the infiltrating bone marrow-derived cells to this process still remains enigmatic.

Improved survival of ischemic cutaneous and musculocutaneous flaps after vascular endothelial growth factor gene transfer using adeno-associated virus vectors.

A major challenge in reconstructive surgery is flap ischemia, which might benefit from induction of therapeutic angiogenesis. Here we demonstrate the effect of an adeno-associated virus (AAV) vector delivering vascular endothelial growth factor (VEGF)165 in two widely recognized in vivo flap models. For the epigastric flap model, animals were injected subcutaneously with 1.5 x 10(11) particles of AAV-VEGF at day 0, 7, or 14 before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF was injected intramuscularly. The delivery of AAV-VEGF significantly improved flap survival in both models, reducing necrosis in all treatment groups compared to controls. The most notable results were obtained by administering the vector 14 days before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF reduced the necrotic area by >50% at 1 week after surgery, with a highly significant improvement in the healing process throughout the following 2 weeks. The therapeutic effect of AAV-VEGF on flap survival was confirmed by histological evidence of neoangiogenesis in the formation of large numbers of CD31-positive capillaries and alpha-smooth muscle actin-positive arteriolae, particularly evident at the border between viable and necrotic tissue. These results underscore the efficacy of VEGF-induced neovascularization for the prevention of tissue ischemia and the improvement of flap survival in reconstructive surgery.

Vascular endothelial growth factor stimulates skeletal muscle regeneration in vivo.

Vascular endothelial growth factor (VEGF) is a major regulator of blood vessel formation during development and in the adult organism. Recent evidence indicates that this factor also plays an important role in sustaining the proliferation and differentiation of different cell types, including progenitor cells of different tissues, including bone marrow, bone, and the central nervous system. Here we show that the delivery of the 165-aa isoform of VEGF-A cDNA using an adeno-associated virus (AAV) vector exerts a powerful effect on skeletal muscle regeneration in vivo. Following ischemia-, glycerol-, or cardiotoxin-induced damage in mouse skeletal muscle, the delivery of AAV-VEGF markedly improved muscle fiber reconstitution with a dose-dependent effect. The expression of both VEGF receptor-1 (VEGFR-1) and VEGFR-2 was upregulated both in the satellite cells of the damaged muscles and during myotube formation in vitro; the VEGF effect was mediated by the VEGFR-2, since the transfer of PlGF, a VEGF family member interacting with the VEGFR-1, was ineffective. These results are consistent with the observation that VEGF promotes the growth of myogenic fibers and protects the myogenic cells from apoptosis in vitro and prompt a therapeutic use for VEGF gene transfer in a variety of muscular disorders.

Potent inhibition of arterial intimal hyperplasia by TIMP1 gene transfer using AAV vectors.

Seminal to the process of arterial restenosis after balloon angioplasty is extracellular matrix degradation by metalloproteinases (MMPs); activity of these proteins is strongly inhibited by the tissue inhibitors of MMPs (TIMPs). Here we exploit gene transfer using an adeno-associated virus (AAV) for TIMP1 gene delivery in a rat model of intimal hyperplasia. High-titer AAV-Timp1 efficiently transduced human coronary artery smooth muscle cells (SMCs) in vitro and inhibited the capacity of these cells to migrate through a Matrigel barrier. In injured rat carotid arteries, AAV vectors were found to transduce SMCs efficiently and to maintain transgene expression for several weeks in vivo. In AAV-Timp1-transduced animals, the intima:media ratio of injured carotids was significantly reduced by 70.5% after 2 weeks, by 58.5% after 1 month, and by 52.4% after 2 months from treatment. The decrease in intimal hyperplasia was paralleled by a significant inhibition of collagen accumulation and by increased elastin deposition in the neointima, two findings that relate to the inhibition of MMP activity. These results indicate that AAV vectors are efficient tools for delivering genes to the arterial wall and emphasize the importance of MMPs for the generation of intimal hyperplasia. Local TIMP1 gene transfer might thus represent an efficient strategy to prevent restenosis.