H19-Dependent Transcriptional Regulation of ß3 and ß4 Integrins Upon Estrogen and Hypoxia Favors Metastatic Potential in Prostate Cancer.

Estrogen and hypoxia promote an aggressive phenotype in prostate cancer (PCa), driving transcription of progression-associated genes. Here, we molecularly dissect the contribution of long non-coding RNA H19 to PCa metastatic potential under combined stimuli, a topic largely uncovered. The effects of estrogen and hypoxia on H19 and cell adhesion molecules' expression were investigated in PCa cells and PCa-derived organotypic slice cultures (OSCs) by qPCR and Western blot. The molecular mechanism was addressed by chromatin immunoprecipitations, overexpression, and silencing assays. PCa cells' metastatic potential was analyzed by in vitro cell-cell adhesion, motility test, and trans-well invasion assay. We found that combined treatment caused a significant H19 down-regulation as compared with hypoxia. In turn, H19 acts as a transcriptional repressor of cell adhesion molecules, as revealed by up-regulation of both ß3 and ß4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and ß integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of ß integrins and invasion was achieved through epigenetic modulation of H19-dependent transcription. Our study revealed that estrogen and hypoxia transcriptionally regulate, via H19, cell adhesion molecules redirecting metastatic dissemination from EMT to a ß integrin-mediated invasion.

SEMA6A/RhoA/YAP axis mediates tumor-stroma interactions and prevents response to dual BRAF/MEK inhibition in BRAF-mutant melanoma.

BACKGROUND: Despite the promise of dual BRAF/MEK inhibition as a therapy for BRAF-mutant (BRAF-mut) melanoma, heterogeneous responses have been observed in patients, thus predictors of benefit from therapy are needed. We have previously identified semaphorin 6A (SEMA6A) as a BRAF-mut-associated protein involved in actin cytoskeleton remodeling. The purpose of the present study is to dissect the role of SEMA6A in the biology of BRAF-mut melanoma, and to explore its predictive potential towards dual BRAF/MEK inhibition. METHODS: SEMA6A expression was assessed by immunohistochemistry in melanoma cohort RECI1 (N = 112) and its prognostic potential was investigated in BRAF-mut melanoma patients from DFCI and TCGA datasets (N = 258). The molecular mechanisms regulated by SEMA6A to sustain tumor aggressiveness and targeted therapy resistance were investigated in vitro by using BRAF-mut and BRAF-wt melanoma cell lines, an inducible SEMA6A silencing cell model and a microenvironment-mimicking fibroblasts-coculturing model. Finally, SEMA6A prediction of benefit from dual BRAF/MEK inhibition was investigated in melanoma cohort RECI2 (N = 14). RESULTS: Our results indicate higher protein expression of SEMA6A in BRAF-mut compared with BRAF-wt melanoma patients and show that SEMA6A is a prognostic indicator in BRAF-mut melanoma from TCGA and DFCI patients cohorts. In BRAF-mut melanoma cells, SEMA6A coordinates actin cytoskeleton remodeling by the RhoA-dependent activation of YAP and dual BRAF/MEK inhibition by dabrafenib+trametinib induces SEMA6A/RhoA/YAP axis. In microenvironment-mimicking co-culture condition, fibroblasts confer to melanoma cells a proliferative stimulus and protect them from targeted therapies, whereas SEMA6A depletion rescues the efficacy of dual BRAF/MEK inhibition. Finally, in BRAF-mut melanoma patients treated with dabrafenib+trametinib, high SEMA6A predicts shorter recurrence-free interval. CONCLUSIONS: Overall, our results indicate that SEMA6A contributes to microenvironment-coordinated evasion of melanoma cells from dual BRAF/MEK inhibition and it might be a good candidate predictor of short-term benefit from dual BRAF/MEK inhibition.

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells.

Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients' outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan-Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.

Corrigendum: Next-Generation Sequencing Approaches for the Identification of Pathognomonic Fusion Transcripts in Sarcomas: The Experience of the Italian ACC Sarcoma Working Group.

[This corrects the article DOI: 10.3389/fonc.2020.00489.].

Next-Generation Sequencing Approaches for the Identification of Pathognomonic Fusion Transcripts in Sarcomas: The Experience of the Italian ACC Sarcoma Working Group.

This work describes the set-up of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.

The alpha6beta4 integrin can regulate ErbB-3 expression: implications for alpha6beta4 signaling and function.

The integrin alpha(6)beta(4) has been shown to facilitate key functions of carcinoma cells, including their ability to migrate, invade, and evade apoptosis. The mechanism involved seems to be a profound effect of alpha(6)beta(4) on specific signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An intimate relationship between alpha(6)beta(4) and growth factor receptors may explain this effect of alpha(6)beta(4) on signaling. Previously, we showed that alpha(6)beta(4) and ErbB-2 can function synergistically to activate the PI3K/Akt pathway. Given that ErbB-2 can activate PI3K only when it heterodimerizes with other members of the epidermal growth factor receptor family, these data imply that other receptors cooperate in this process. Here, we report that alpha(6)beta(4) can regulate the expression of ErbB-3 using several different models and that the consequent formation of an ErbB-2/ErbB-3 heterodimer promotes the alpha(6)beta(4)-dependent activation of PI3K/Akt and the ability of this integrin to impede apoptosis of carcinoma cells. Our data also support the hypothesis that alpha(6)beta(4) can regulate ErbB-3 expression at the translational level as evidenced by the findings that alpha(6)beta(4) does not increase ErbB-3 mRNA significantly, and that this regulation is both rapamycin sensitive and dependent on eukaryotic translation initiation factor 4E. These findings provide one mechanism to account for the activation of PI3K by alpha(6)beta(4) and they also provide insight into the regulation of ErbB-3 in carcinoma cells.

The prognostic significance of positive peritoneal cytology in endometrial cancer and its correlations with L1-CAM biomarker.

BACKGROUND: The aim of this study was to evaluate the prognostic role of positive peritoneal cytology (PPC) in a cohort of patients with endometrial cancer (EC). The secondary objective was to correlate the PPC and the expression of L1CAM in a group of patients with recurrence endometrial disease. METHODS: All women diagnosed with EC and who performed a peritoneal cytology at "Regina Elena" National Cancer Institute of Rome from 2001 to 2013 were included in the study. Patients were divided into two groups according to positivity at peritoneal cytology. Moreover, patients with a recurrence disease and whose a tissue microarray (TMA) tumor sample was available underwent a L1CAM analysis. RESULTS: Seven hundred sixty six patients underwent to EC staging in our Institute: 696 (90.8%) with negative and 70 (9.2%) with positive cytology. Five-year recurrence rate was higher in women with PPC (46.9% vs 18.4%, p = 0 < 0.0001) and, in particular, distant recurrence (86.7% vs 53.4%, p = 0.03). Moreover, we found an interesting pattern of recurrence disease in the group of early stage of EC with NPC and positive L1CAM. CONCLUSIONS: Our results support the data that PPC may be a potential prognostic factor in early EC, due to its significant association with other risk factors and its significant influence on survival. Our findings confirm the need for large studies that point out the role of PPC and new prognostic factors, including biomarkers as L1CAM.

Very low intensity ultrasounds as a new strategy to improve selective delivery of nanoparticles-complexes in cancer cells.

BACKGROUND: The possibility to combine Low Intensity UltraSound (LIUS) and Nanoparticles (NP) could represent a promising strategy for drugs delivery in tumors difficult to treat overcoming resistance to therapies. On one side the NP can carry drugs that specifically target the tumors on the other the LIUS can facilitate and direct the delivery to the tumor cells. In this study, we investigated whether Very Low Intensity UltraSound (VLIUS), at intensities lower than 120 mW/cm2, might constitute a novel strategy to improve delivery to tumor cells. Thus, in order to verify the efficacy of this novel modality in terms of increase selective uptake in tumoral cells and translate speedily in clinical practice, we investigated VLIUS in three different in vitro experimental tumor models and normal cells adopting three different therapeutic strategies. METHODS: VLIUS at different intensities and exposure time were applied to tumor and normal cells to evaluate the efficiency in uptake of labeled human ferritin (HFt)-based NP, the delivery of NP complexed Firefly luciferase reported gene (lipoplex-LUC), and the tumor-killing of chemotherapeutic agent. RESULTS: Specifically, we found that specific VLIUS intensity (120 mW/cm2) increases tumor cell uptake of HFt-based NPs at specific concentration (0.5 mg/ml). Similarly, VLIUS treatments increase significantly tumor cells delivery of lipoplex-LUC cargos. Furthermore, of interest, VLIUS increases tumor killing of chemotherapy drug trabectedin in a time dependent fashion. Noteworthy, VLIUS treatments are well tolerated in normal cells with not significant effects on cell survival, NPs delivery and drug-induced toxicity, suggesting a tumor specific fashion. CONCLUSIONS: Our data shed novel lights on the potential application of VLIUS for the design and development of novel therapeutic strategies aiming to efficiently deliver NP loaded cargos or anticancer drugs into more aggressive and unresponsive tumors niche.

Histone deacetylase inhibitor ITF2357 leads to apoptosis and enhances doxorubicin cytotoxicity in preclinical models of human sarcoma.

Sarcomas are rare tumors with generally poor prognosis, for which current therapies have shown limited efficacy. Histone deacetylase inhibitors (HDACi) are emerging anti-tumor agents; however, little is known about their effect in sarcomas. By using established and patient-derived sarcoma cells with different subtypes, we showed that the pan-HDACi, ITF2357, potently inhibited in vitro survival in a p53-independent manner. ITF2357-mediated cell death implied the activation of mitochondrial apoptosis, as attested by induction of pro-apoptotic BH3-only proteins and a caspases-dependent mechanism. ITF2357 also induced autophagy, which protected sarcoma cells from apoptotic cell death. ITF2357 activated forkhead box (FOXO) 1 and 3a transcription factors and their downstream target genes, however, silencing of both FOXO1 and 3a did not protect sarcoma cells against ITF2357-induced apoptosis and upregulated FOXO4 and 6. Notably, ITF2357 synergized with Doxorubicin to induce cell death of established and patient-derived sarcoma cells. Furthermore, combination treatment strongly impaired xenograft tumor growth in vivo, when compared to single treatments, suggesting that combination of ITF2357 with Doxorubicin has the potential to enhance sensitization in different preclinical models of sarcoma. Overall, our study highlights the therapeutic potential of ITF2357, alone or in rational combination therapies, for bone and soft tissue sarcomas management.

Endometrial cancer prognosis correlates with the expression of L1CAM and miR34a biomarkers.

BACKGROUND: Patients with endometrial cancer (EC) and presumably with good prognosis may develop a recurrence indicating that the classification of this tumor is still not definitive and that new markers are needed to identify a subgroup at risk of relapse. The cell adhesion molecule L1CAM is highly expressed in several human carcinomas and has recently been described as a new marker for endometrial and ovarian carcinomas. The aim of this study was to determine the relevance of L1CAM in recurrent EC. METHODS: In this work we have analyzed, by immunohistochemical and RT-qPCR analysis, the expression of L1CAM in a cohort of 113 endometrial cancers at different stages, which 50% have relapsed. As a predictor of good outcome, the tumors were also analyzed for the expression of miR-34a, a post-transcriptional regulator of L1CAM. RESULTS: Among metastatic EC, the highest levels (60%) and the median level (24%) of L1CAM in tumors correlate with the progression, suggesting that the expression of this molecule is linked to the tumor component most involved in metastatic processes. We also found an inverse correlation between miR-34a and L1CAM protein expression, suggesting that miR-34a is a positive prognostic marker of EC. CONCLUSIONS: Our results demonstrate the expression of L1CAM and miR-34a in EC as prognostic factors that identify subgroup of patients at high risk of recurrence suggesting for them more aggressive schedules of treatment.

HMGA1/E2F1 axis and NFkB pathways regulate LPS progression and trabectedin resistance.

Although the medical treatments of sarcoma have evolved in the last years, a significant portion of patients develops recurrence after therapies suggesting the need to identify novel targets to improve the treatments. By the use of patient-derived and established cell lines from liposarcoma, as well as specimens from patient biopsies, we found that HMGA1 is involved in the progression of dedifferentiated and myxoid liposarcoma. The immunohistochemical and RT-PCR analyses of 68 liposarcoma specimens revealed a significant high expression of HMGA1, at the protein and RNA levels, both in myxoid and dedifferentiated liposarcoma subtypes compared with differentiated ones. Loss- and gain-of-function experiments by HMGA1-specific depletion and overexpression in dedifferentiated and myxoid liposarcoma cells showed the contribution of this oncogenic factor in cell proliferation, motility, invasion, and drug resistance. The in vitro and in vivo treatment of myxoid liposarcoma with trabectedin, a drug with a potent anti-tumor activity, revealed downregulation of HMGA1, E2F1, and its-downstream targets, vimentin and ZEB1, indicating a critical role of trabectedin in inhibiting the mesenchymal markers of these tumors through the HMGA1/E2F1 axis. These data were also confirmed in patients' tumor biopsies being HMGA1, E2F1, and vimentin expression significantly reduced upon trabectedin therapy, administered as neo-adjuvant chemotherapy. Furthermore, trabectedin treatment inhibits in vitro NFkB pathway in mixoyd liposarcoma sensitive but not in resistant counterparts, and the inhibition of NFkB pathway re-sensitizes the resistant cells to trabectedin treatment. These data support the rational for combining NFkB inhibitors with trabectedin in liposarcoma patients, who have become resistant to the drug.

Involvement of alpha6beta4 integrin in the mechanisms that regulate breast cancer progression.

Integrin alpha6beta4 is mostly expressed in epithelial tissues and endothelial and Schwann cells. Expression of alpha6beta4 is increased in many epithelial tumours, implicating its involvement in tumour malignancy. Moreover, this integrin activates several key signalling molecules in carcinoma cells, but its ability to activate the phosphatidylinositol 3-kinase/Akt pathway is among the mechanisms by which alpha6beta4 integrin regulates tumour behaviour. In this review we discuss the biological and clinical features of alpha6beta4 integrin that allow it to promote tumour survival and progression of mammary tumours.

Loss of beta4 integrin subunit reduces the tumorigenicity of MCF7 mammary cells and causes apoptosis upon hormone deprivation.

PURPOSE: The alpha6beta4 integrin, a laminin receptor, has been implicated from many studies in tumor progression and invasion. We showed that the beta4 integrin subunit associates with the ErbB-2 tyrosine kinase in human mammary carcinoma cell lines and that its overexpression in NIH3T3/ErbB-2-transformed cells causes a constitutive activation of phosphatidylinositol 3-kinase (PI3K), inducing a strong increase of their invasive capacity. In this study, we investigated the biological consequences of interference with the endogenous beta4 integrin subunit expression. EXPERIMENTAL DESIGN: In vitro and in vivo tumor growth and the biochemical consequences of beta4 integrin inactivation were studied in mammary tumor cells by using short hairpin RNA approach. RESULTS: Our data show that tumor growth of mammary tumor cells strictly depends on beta4 expression, confirming the relevance of beta4 protein in these cells. Moreover, interference with beta4 expression significantly reduces endogenous PI3K activity and AKT and mammalian target of rapamycin phosphorylation. Accordingly, with these results and considering that PI3K activity in mammary tumor plays a relevant role in hormone resistance, we asked whether beta4 expression might be relevant for hormone responsiveness in these cells. Data reported indicate that the interference with endogenous beta4 expression, upon hormone deprivation, induces caspase-9 and cytochrome c-mediated apoptosis, which is enhanced upon tamoxifen treatment. On the other hand, the expression of myr-AKT in MCF7 beta4-short hairpin RNA cells rescues the cells from apoptosis in the absence of hormones and upon tamoxifen treatment. CONCLUSIONS: Overall, these results confirm the relevance of beta4 expression in mammary tumors and indicate this integrin as a relevant target for tumor therapy.

Homeodomain-interacting protein kinase-2 restrains cytosolic phospholipase A2-dependent prostaglandin E2 generation in human colorectal cancer cells.

PURPOSE: Homeodomain-interacting protein kinase-2 (HIPK2), a corepressor for homeodomain transcription factors, is a multifunctional kinase whose role in tumor cell survival is not completely clarified. We addressed whether HIPK2 restrains colon tumorigenesis by turning off cytosolic phospholipase A2 (cPLA2)-dependent prostaglandin E2 (PGE2) generation in the light of overwhelming evidence suggesting the contribution of this prostanoid in a variety of cancers. EXPERIMENTAL DESIGN: In the human colorectal cancer cell line, RKO, we studied the effect of RNA interference for HIPK2 (HIPK2i) on prostanoid biosynthesis, both in the absence and in the presence of the cPLA2 inhibitor arachidonyl trifluoromethyl ketone. We evaluated the role of HIPK2 in the cPLA2 gene regulation by reverse transcriptase-PCR, transcriptional activity, and chromatin immunoprecipitation analyses. The involvement of HIPK2 in tumorigenicity in vivo was studied by tumor growth of HIPK2i cells in nude mice. We compared the gene expression of HIPK2 and cPLA2 in human colorectal cancer specimens by reverse transcriptase-PCR. RESULTS: HIPK2 silencing was associated with rousing PGE2 biosynthesis that was profoundly suppressed by the cPLA2 inhibitor. HIPK2 overexpression, along with histone deacetylase-1, inhibited the cPLA2-luc promoter that is strongly acetylated in HIPK2i cells. The tumors derived from HIPK2i cells injected in nude mice showed noticeably increased growth compared with parental cells. HIPK2 mRNA levels were significantly higher in colorectal cancers of patients with familial adenomatous polyposis, which showed undetectable cPLA2 levels compared with sporadic colorectal cancer expressing cPLA2. CONCLUSIONS: Our findings reveal the novel mechanism of HIPK2 to restrain progression of human colon tumorigenesis, at least in part, by turning off cPLA2-dependent PGE2 generation.

Prognostic role of NF-YA splicing isoforms and Lamin A status in low grade endometrial cancer.

Although most cases of low grade (G1) endometrial cancer (EC) do not behave aggressively, in rare instances, can progress in a highly aggressive manner. In this study we analyzed formalin-fixed, paraffin-embedded (FFPE) EC tissues to find novel clinical and biological features to help diagnosis and treatment of G1 ECs s in order to better stratify patient risk of recurrence. A retrospective cohort of FFPE specimens from patients with EC (n=87) and benign tissue specimens (NE) from patients who underwent a hysterectomy to treat other benign disease (n = 13) were collected. Total RNA and proteins were extracted and analyzed, respectively, by quantitative PCR and western blotting. NF-YAs is expressed and lamin A is down-modulated in all high grade (G2 and G3) ECs. In G1 ECs, NF-YAs expression is heterogeneous being expressed only in a subset of these tumours. Interestingly, the G1 ECs that express NF-YAs display low levels of lamin A similar to those present in G2 and G3 ECs. Of note, this pattern of NF-YAs and lamin A expression correlates with tumor aggressiveness assessed by comparative analysis with estrogen receptor (ER) status and epithelial-mesenchymal transition (EMT) markers thus suggesting its potential role as biomarker of tumour aggressiveness in G1 EC. In all grade ECs, lamin A is strongly downmodulated, being its expression inversely correlated with tumor aggressiveness and its loss of expression. We identified NF-YAs and lamin A expression levels as novel potential biomarkers useful to identify G1 ECs patients with risk of recurrence.

Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers.

Although the medical treatment of colorectal cancer has evolved greatly in the last years, a significant portion of early-stage patients develops recurrence after therapies. The current clinical trials are directed to evaluate new drug combinations and treatment schedules. By the use of patient-derived or established colon cancer cell lines, we found that the tyrosine kinase receptor HER3 is involved in the mechanisms of resistance to therapies. In agreement, the immunohistochemical analysis of total and phospho-HER3 expression in 185 colorectal cancer specimens revealed a significant correlation with lower disease-free survival. Targeting HER3 by the use of the monoclonal antibody patritumab we found induction of growth arrest in all cell lines. Despite the high efficiency of patritumab in abrogating the HER3-dependent activation of PI3K pathway, the HER2 and EGFR-dependent MAPK pathway is activated as a compensatory mechanism. Interestingly, we found that the MEK-inhibitor trametinib inhibits, as expected, the MAPK pathway but induces the HER3-dependent activation of PI3K pathway. The combined treatment results in the abrogation of both PI3K and MAPK pathways and in a significant reduction of cell proliferation and survival. These data suggest a new strategy of therapy for HER3-overexpressing colon cancers.

Resensitization of breast cancer cells to anoikis by tropomyosin-1: role of Rho kinase-dependent cytoskeleton and adhesion.

Two most common properties of malignant cells are the presence of aberrant actin cytoskeleton and resistance to anoikis. Suppression of several key cytoskeletal proteins, including tropomyosin-1 (TM1), during neoplastic transformation is hypothesized to contribute to the altered cytoskeleton and neoplastic phenotype. Using TM1 as a paradigm, we have shown that cytoskeletal proteins induce anoikis in breast cancer (MCF-7 and MDA MB 231) cells. Here, we have tested the hypothesis that TM1-mediated cytoskeletal changes regulate integrin activity and the sensitivity to anoikis. TM1 expression in MDA MB 231 cells promotes the assembly of stress fibers, induces rapid anoikis via caspase-dependent pathways involving the release of cytochrome c. Further, TM1 inhibits binding of MDA MB 231 cells to collagen I, but promotes adhesion to laminin. Inhibition of Rho kinase disrupts TM1-mediated cytoskeletal reorganization and adhesion to the extracellular matrix components, whereas the parental cells attach to collagen I, spread and form extensive actin meshwork in the presence of Rho kinase inhibitor, underscoring the differences in parental and TM1-transduced breast cancer cells. Further, treatment with the cytoskeletal disrupting drugs rescues the cells from TM1-induced anoikis. These new findings demonstrate that the aberrant cytoskeleton contributes to neoplastic transformation by conferring resistance to anoikis. Restoration of stress fiber network through enhanced expression of key cytoskeletal proteins may modulate the activity of focal adhesions and sensitize the neoplastic cells to anoikis.

Unacylated ghrelin induces oxidative stress resistance in a glucose intolerance and peripheral artery disease mouse model by restoring endothelial cell miR-126 expression.

Reactive oxygen species (ROS) are crucial in long-term diabetes complications, including peripheral artery disease (PAD). In this study, we have investigated the potential clinical impact of unacylated ghrelin (UnAG) in a glucose intolerance and PAD mouse model. We demonstrate that UnAG is able to protect skeletal muscle and endothelial cells (ECs) from ROS imbalance in hind limb ischemia-subjected ob/ob mice. This effect translates into reductions in hind limb functional impairment. We show that UnAG rescues sirtuin 1 (SIRT1) activity and superoxide dismutase-2 (SOD-2) expression in ECs. This leads to SIRT1-mediated p53 and histone 3 lysate 56 deacetylation and results in reduced EC senescence in vivo. We demonstrate, using small interfering RNA technology, that SIRT1 is also crucial for SOD-2 expression. UnAG also renews micro-RNA (miR)-126 expression, resulting in the posttranscriptional regulation of vascular cell adhesion molecule 1 expression and a reduced number of infiltrating inflammatory cells in vivo. Loss-of-function experiments that target miR-126 demonstrate that miR-126 also controls SIRT1 and SOD-2 expression, thus confirming its role in driving UnAG-mediated EC protection against ROS imbalance. These results indicate that UnAG protects vessels from ROS imbalance in ob/ob mice by rescuing miR-126 expression, thus emphasizing its potential clinical impact in avoiding limb loss in PAD.

ATM kinase sustains HER2 tumorigenicity in breast cancer.

ATM kinase preserves genomic stability by acting as a tumour suppressor. However, its identification as a component of several signalling networks suggests a dualism for ATM in cancer. Here we report that ATM expression and activity promotes HER2-dependent tumorigenicity in vitro and in vivo. We reveal a correlation between ATM activation and the reduced time to recurrence in patients diagnosed with invasive HER2-positive breast cancer. Furthermore, we identify ATM as a novel modulator of HER2 protein stability that acts by promoting a complex of HER2 with the chaperone HSP90, therefore preventing HER2 ubiquitination and degradation. As a consequence, ATM sustains AKT activation downstream of HER2 and may modulate the response to therapeutic approaches, suggesting that the status of ATM activity may be informative for the treatment and prognosis of HER2-positive tumours. Our findings provide evidence for ATM's tumorigenic potential revising the canonical role of ATM as a pure tumour suppressor.

Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells.

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.