Co-immunoprecipitation of Protein Complexes from Different Subcellular Compartments in Vasculogenic Mimicry Studies.

Aberrant extravascular expression of VE-cadherin has been observed in metastasis associated with vasculogenic mimicry (VM); we have recently shown that in VM prone cells VE-cadherin (mainly in the form of phospho-VE-cadherin in Y658) is in part located in the cell nucleus, which associates with p120-catenin and the transcription factor kaiso allowing increased plasticity that potentiates VM development in malignant cells. In this chapter, we describe the protocol to analyze protein-protein interactions in subcellular fractions with particular focus in VE-cadherin. The verification of the subcellular interactome of VE-cadherin and other key proteins involved in VM shed light to novel functions of endothelial proteins aberrantly expressed in tumor cells and their consequences in cell plasticity during VM development.

The PARP Inhibitor Olaparib Modulates the Transcriptional Regulatory Networks of Long Non-Coding RNAs during Vasculogenic Mimicry.

In highly metastatic tumors, vasculogenic mimicry (VM) involves the acquisition by tumor cells of endothelial-like traits. Poly-(ADP-ribose) polymerase (PARP) inhibitors are currently used against tumors displaying BRCA1/2-dependent deficient homologous recombination, and they may have antimetastatic activity. Long non-coding RNAs (lncRNAs) are emerging as key species-specific regulators of cellular and disease processes. To evaluate the impact of olaparib treatment in the context of non-coding RNA, we have analyzed the expression of lncRNA after performing unbiased whole-transcriptome profiling of human uveal melanoma cells cultured to form VM. RNAseq revealed that the non-coding transcriptomic landscape differed between olaparib-treated and non-treated cells: olaparib significantly modulated the expression of 20 lncRNAs, 11 lncRNAs being upregulated, and 9 downregulated. We subjected the data to different bioinformatics tools and analysis in public databases. We found that copy-number variation alterations in some olaparib-modulated lncRNAs had a statistically significant correlation with alterations in some key tumor suppressor genes. Furthermore, the lncRNAs that were modulated by olaparib appeared to be regulated by common transcription factors: ETS1 had high-score binding sites in the promoters of all olaparib upregulated lncRNAs, while MZF1, RHOXF1 and NR2C2 had high-score binding sites in the promoters of all olaparib downregulated lncRNAs. Finally, we predicted that olaparib-modulated lncRNAs could further regulate several transcription factors and their subsequent target genes in melanoma, suggesting that olaparib may trigger a major shift in gene expression mediated by the regulation lncRNA. Globally, olaparib changed the lncRNA expression landscape during VM affecting angiogenesis-related genes.

The Multifactorial Role of PARP-1 in Tumor Microenvironment.

Poly(ADP-ribose) polymerases (PARPs), represent a family of 17 proteins implicated in a variety of cell functions; some of them possess the enzymatic ability to synthesize and attach poly (ADP-ribose) (also known as PAR) to different protein substrates by a post-translational modification; PARPs are key components in the cellular response to stress with consequences for different physiological and pathological events, especially during neoplasia. In recent years, using PARP inhibitors as antitumor agents has raised new challenges in understanding their role in tumor biology. Notably, the function of PARPs and PAR in the dynamic of tumor microenvironment is only starting to be understood. In this review, we summarized the conclusions arising from recent studies on the interaction between PARPs, PAR and key features of tumor microenvironment such as hypoxia, autophagy, tumor initiating cells, angiogenesis and cancer-associated immune response.

Enhancing the Bystander and Abscopal Effects to Improve Radiotherapy Outcomes.

In this paper, we summarize published articles and experiences related to the attempt to improve radiotherapy outcomes and, thus, to personalize the radiation treatment according to the individual characteristics of each patient. The evolution of ideas and the study of successively published data have led us to envisage new biophysical models for the interpretation of tumor and healthy normal tissue response to radiation. In the development of the model, we have shown that when mesenchymal stem cells (MSCs) and radiotherapy are administered simultaneously in experimental radiotherapy on xenotumors implanted in a murine model, the results of the treatment show the existence of a synergic mechanism that is able to enhance the local and systemic actions of the radiation both on the treated tumor and on its possible metastasis. We are convinced that, due to the physical hallmarks that characterize the neoplastic tissues, the physical-chemical tropism of MSCs, and the widespread functions of macromolecules, proteins, and exosomes released from activated MSCs, the combination of radiotherapy plus MSCs used intratumorally has the effect of counteracting the pro-tumorigenic and pro-metastatic signals that contribute to the growth, spread, and resistance of the tumor cells. Therefore, we have concluded that MSCs are appropriate for therapeutic use in a clinical trial for rectal cancer combined with radiotherapy, which we are going to start in the near future.

Human mesenchymal stem cells enhance the systemic effects of radiotherapy.

The outcome of radiotherapy treatment might be further improved by a better understanding of individual variations in tumor radiosensitivity and normal tissue reactions, including the bystander effect. For many tumors, however, a definitive cure cannot be achieved, despite the availablity of more and more effective cancer treatments. Therefore, any improvement in the efficacy of radiotherapy will undoubtedly benefit a significant number of patients. Many experimental studies measure a bystander component of tumor cell death after radiotherapy, which highlights the importance of confirming these observations in a preclinical situation. Mesenchymal stem cells (MSCs) have been investigated for use in the treatment of cancers as they are able to both preferentially home onto tumors and become incorporated into their stroma. This process increases after radiation therapy. In our study we show that in vitro MSCs, when activated with a low dose of radiation, are a source of anti-tumor cytokines that decrease the proliferative activity of tumor cells, producing a potent cytotoxic synergistic effect on tumor cells. In vivo administration of unirradiated mesenchymal cells together with radiation leads to an increased efficacy of radiotherapy, thus leading to an enhancement of short and long range bystander effects on primary-irradiated tumors and distant-non-irradiated tumors. Our experiments indicate an increased cell loss rate and the decrease in the tumor cell proliferation activity as the major mechanisms underlying the delayed tumor growth and are a strong indicator of the synergistic effect between RT and MSC when they are applied together for tumor treatment in this model.

Direct and bystander radiation effects: a biophysical model and clinical perspectives.

In planning treatment for each new patient, radiation oncologists pay attention to the aspects that they control. Thus their attention is usually focused on volume and dose. The dilemma for the physician is how to protract the treatment in a way that maximizes control of the tumor and minimizes normal tissue injury. The initial radiation-induced damage to DNA may be a biological indicator of the quantity of energy transferred to the DNA. However, until now the biophysical models proposed cannot explain either the early or the late adverse effects of radiation, and a more general theory appears to be required. The bystander component of tumor cell death after radiotherapy measured in many experimental works highlights the importance of confirming these observations in a clinical situation.

Poly(ADP-ribose) signaling in cell death.

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.

Growth and spontaneous differentiation of umbilical-cord stromal stem cells on activated carbon cloth.

We have investigated the capacity of activated carbon cloth to support the growth and differentiation of human mesenchymal umbilical-cord stromal stem cells. Our results demonstrate that this scaffold provides suitable conditions for the development of cell-derived matrix proteins and facilitates the growth of undifferentiated stem cells with the ability to induce osteogenic and chondrogenic differentiation. Immunoflourescence staining revealed extensive expression of collagen in all the samples, and collagen type II and osteopontin within the samples cultivated in specific differentiation-inducing media. Cell growth and the formation of natural collagen, calcium-magnesium carbonate and hydroxyapatite crystals, together with the self-assemblage of collagen to produce suprafibrillar arrangements of fibrils all occur simultaneously and can be studied together ex vivo under physiological conditions. Furthermore, the spontaneous differentiation of stem cells cultured on activated carbon cloth with no osteogenic supplements opens up new possibilities for bone-tumour engineering and treatment of traumatic and degenerative bone diseases.

The importance of bystander effects in radiation therapy in melanoma skin-cancer cells and umbilical-cord stromal stem cells.

PURPOSE: To examine direct and bystander radiation-induced effects in normal umbilical-cord stromal stem cell (HCSSC) lines and in human cancer cells. MATERIALS AND METHODS: The UCSSC lines used in this study were obtained in our laboratory. Two cell lines (UCSSC 35 and UCSSC 37) and two human melanoma skin-cancer cells (A375 and G361) were exposed to ionizing radiation to measure acute radiation-dosage cell-survival curves and radiation-induced bystander cell-death response. Normal cells, although extremely sensitive to ionizing radiation, were resistant to the bystander effect whilst tumor cells were sensitive to irradiated cell-conditioned media, showing a dose-response relationship that became saturated at relatively low doses. We applied a biophysical model to describe bystander cell-death through the binding of a ligand to the cells. This model allowed us to calculate the maximum cell death (χ(max)) produced by the bystander effect together with its association constant (K(By)) in terms of dose equivalence (Gy). The values obtained for K(By) in A375 and G361 cells were 0.23 and 0.29 Gy, respectively. CONCLUSION: Our findings help to understand how anticancer therapy could have an additional decisive effect in that the response of sub-lethally hit tumor cells to damage might be required for therapy to be successful because the survival of cells communicating with irradiated cells is reduced.

Hypermethylated 14-3-3-sigma and ESR1 gene promoters in serum as candidate biomarkers for the diagnosis and treatment efficacy of breast cancer metastasis.

BACKGROUND: Numerous hypermethylated genes have been reported in breast cancer, and the silencing of these genes plays an important role in carcinogenesis, tumor progression and diagnosis. These hypermethylated promoters are very rarely found in normal breast. It has been suggested that aberrant hypermethylation may be useful as a biomarker, with implications for breast cancer etiology, diagnosis, and management. The relationship between primary neoplasm and metastasis remains largely unknown. There has been no comprehensive comparative study on the clinical usefulness of tumor-associated methylated DNA biomarkers in primary breast carcinoma and metastatic breast carcinoma. The objective of the present study was to investigate the association between clinical extension of breast cancer and methylation status of estrogen receptor1 (ESR1) and stratifin (14-3-3-sigma) gene promoters in disease-free and metastatic breast cancer patients. METHODS: We studied two cohorts of patients: 77 patients treated for breast cancer with no signs of disease, and 34 patients with metastatic breast cancer. DNA was obtained from serum samples, and promoter methylation status was determined by using DNA bisulfite modification and quantitative methylation-specific PCR. RESULTS: Serum levels of methylated gene promoter 14-3-3-sigma significantly differed between Control and Metastatic Breast Cancer groups (P < 0.001), and between Disease-Free and Metastatic Breast Cancer groups (P < 0.001). The ratio of the 14-3-3-sigma level before the first chemotherapy cycle to the level just before administration of the second chemotherapy cycle was defined as the Biomarker Response Ratio [BRR]. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups. Subsequent ROC analysis showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39. The area under the ROC curve (Z = 0.804 +/- 0.074) indicates that this test is a good approach to post-treatment prognosis. CONCLUSIONS: The relationship of 14-3-3-sigma with breast cancer metastasis and progression found in this study suggests a possible application of 14-3-3-sigma as a biomarker to screen for metastasis and to follow up patients treated for metastatic breast cancer, monitoring their disease status and treatment response.

Inhibition of poly adenosine diphosphate-ribose polymerase decreases hepatocellular carcinoma growth by modulation of tumor-related gene expression.

UNLABELLED: Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to a lack of effective treatment options. In HCC a significant role is played by DNA damage and the inflammatory response. Poly (ADP-ribose) polymerase-1 (PARP-1) is an important protein that regulates both these mechanisms. The objective of this study was to examine the effect of pharmacology PARP-1 inhibition on the reduction of tumor volume of HCC xenograft and on the hepatocarcinogenesis induced by diethyl-nitrosamine (DEN). Pharmacologic PARP-1 inhibition with DPQ greatly reduces tumor xenograft volume with regard to a nontreated xenograft (394 mm(3) versus 2,942 mm(3), P < 0.05). This observation was paralleled by reductions in xenograft mitosis (P = 0.02) and tumor vasculogenesis (P = 0.007, confirmed by in vitro angiogenesis study), as well as by an increase in the number of apoptotic cells in DPQ-treated mice (P = 0.04). A substantial difference in key tumor-related gene expression (transformed 3T3 cell double minute 2 [MDM2], FLT1 [vascular endothelial growth factor receptor-1, VEGFR1], epidermal growth factor receptor [EPAS1]/hypoxia-inducible factor 2 [HIF2A], EGLN1 [PHD2], epidermal growth factor receptor [EGFR], MYC, JUND, SPP1 [OPN], hepatocyte growth factor [HGF]) was found between the control tumor xenografts and the PARP inhibitor-treated xenografts (data confirmed in HCC cell lines using PARP inhibitors and PARP-1 small interfering RNA [siRNA]). Furthermore, the results obtained in mice treated with DEN to induce hepatocarcinogenesis showed, after treatment with a PARP inhibitor (DPQ), a significant reduction both in preneoplastic foci and in the expression of preneoplastic markers and proinflammatory genes (Gstm3, Vegf, Spp1 [Opn], IL6, IL1b, and Tnf), bromodeoxyuridine incorporation, and NF-kappaB activation in the initial steps of carcinogenesis (P < 0.05). CONCLUSION: This study shows that PARP inhibition is capable of controlling HCC growth and preventing tumor vasculogenesis by regulating the activation of different genes involved in tumor progression.

Quantitative detection of methylated ESR1 and 14-3-3-sigma gene promoters in serum as candidate biomarkers for diagnosis of breast cancer and evaluation of treatment efficacy.

The aim of the present study was to investigate the association between gene hypermethylation and main clinicopathological features of breast cancer, including diagnosis and treatment response. A sensitive SYBR green methylation-specific PCR technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of ESR1, APC, RARB, 14-3-3-sigma and E-cad gene promoter regions as breast cancer biomarkers. Analyses were conducted of preoperative sera from 106 women with breast cancer, 34 with benign breast disease and 74 with no evidence of breast disease and of post-treatment sera from 60 of the breast cancer patients. Mean serum values of methylated ESR1 and 14-3-3-sigma gene promoters significantly differed between breast cancer patients and healthy controls (p = 0.0112 for ESR1 and p = 0.0047 for 14-3-3-sigma). When their results were combined, it was found that hypermethylation of these two genes differentiated between breast cancer patients and healthy controls (p < 0.0001) with a sensitivity of 81% (95% confidence interval: 72-88%) and specificity of 88% (95% CI: 78-94%). Presence of methylated ESR1 in serum of breast cancer patients was associated with the ER negative phenotype (p = 0.0179). Serum hypermethylation at ESR1 and 14-3-3-sigma loci was observed in cancer patients, in situ carcinoma and benign breast disease. No significant differences in methylated ERS1 or 14-3-3-sigma values were observed between pre-surgery and post-treatment measurements. Preliminary clinical applications of this approach have revealed several shortcomings, including a frequent presence of methylated 14-3-3-sigma in sera from women with breast benign disease. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA remains a promising tool for cancer risk assessment.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes.

INTRODUCTION: Radiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects. METHODS: Intrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments. RESULTS: Radiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects. CONCLUSION: After radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Transcription regulation of TNF-alpha-early response genes by poly(ADP-ribose) polymerase-1 in murine heart endothelial cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) has been involved in endothelial cell dysfunction associated with various pathophysiological conditions. The intrinsic mechanism of PARP-1-mediated endothelial cell dysfunction could be related to PARP-1 overactivation, NAD(+) consumption and ATP depletion. An alternative way could involve transcription regulation. By using high-density microarrays, we examined early tumor necrosis factor alpha (TNF-alpha)-stimulated gene expression profiles in PARP-1(+/+) and PARP-1(-/-) murine heart endothelial cells. TNF-alpha modulated a significant number of genes in both cell types. We have identified a set of genes whose expression in response to TNF-alpha is modulated by PARP-1, whereas the expression of others is PARP-1-independent. Up-regulation of several genes involved in the inflammatory response is hampered in the absence of PARP-1. Moreover, NF-kappaB-dependent transcriptional activation is partially inhibited in PARP-1(-/-) compared to PARP-1(+/+) cells. However, we found that PARP-1 might also silence transcription of several NF-kappaB target genes. Overall, our results show that PARP-1 is regulating the expression of genes by the endothelial cells both in a positive and a negative fashion, with the final effects depending on the gene. Individual studies of these genes are now necessary to clarify the intrinsic mechanism by which PARP-1 is controlling transcription and thereby finding out different therapeutic approaches involving PARP-1.