Astrocytes promote progression of breast cancer metastases to the brain via a KISS1-mediated autophagy.

Formation of metastases, also known as cancer dissemination, is an important stage of breast cancer (BrCa) development. KISS1 expression is associated with inhibition of metastases development. Recently we have demonstrated that BrCa metastases to the brain exhibit low levels of KISS1 expression at both mRNA and protein levels. By using multicolor immunofluorescence and coculture techniques here we show that normal adult astrocytes in the brain are capable of promoting metastatic transformation of circulating breast cancer cells localized to the brain through secretion of chemokine CXCL12. The latter was found in this study to downregulate KISS1 expression at the post-transcriptional level via induction of microRNA-345 (MIR345). Furthermore, we demonstrated that ectopic expression of KISS1 downregulates ATG5 and ATG7, 2 key modulators of autophagy, and works concurrently with autophagy inhibitors, thereby implicating autophagy in the mechanism of KISS1-mediated BrCa metastatic transformation. We also found that expression of KISS1 in human breast tumor specimens inversely correlates with that of MMP9 and IL8, implicated in the mechanism of metastatic invasion, thereby supporting the role of KISS1 as a potential regulator of BrCa metastatic invasion in the brain. This conclusion is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-MIR345- KISS1 axis.

CMV70-3P miRNA contributes to the CMV mediated glioma stemness and represents a target for glioma experimental therapy.

Glioblastoma multiforme (GBM) is a rapidly progressive brain tumor with a median survival of 15-19 months. Therapeutic resistance and recurrence of the disease is attributed to cancer stem cells (CSC). Here, we report that CMV70-3P miRNA encoded by CMV increases GBM CSC stemness. Inhibition of CMV70-3P expression using oligo inhibitors significantly attenuated the ability of primary glioma cells to proliferate and form neurospheres. At the molecular level, we show that CM70-3P increases expression of cellular SOX2. Collectively, these findings indicate that CMV70-3P is a potential regulator of CMV- mediated glioma progression and cancer stemness.

MT1-MMP silencing by an shRNA-armed glioma-targeted conditionally replicative adenovirus (CRAd) improves its anti-glioma efficacy in vitro and in vivo.

MMP14 (MT1-MMP) is a cell membrane-associated proteinase of the extracellular matrix, whose biological roles vary from angiogenesis to cell proliferation and survival. We recently found a direct correlation between MMP14 expression levels in brain tumors of glioma patients and the disease progression. By using gene silencing as an experimental approach we found that MMP14 knockdown decreases production of pro-angiogenic factors such as VEGF and IL8 and thereby suppresses angiogenesis in glioma tumors. Although the clinical relevance of MMP14 down-regulation and its possible implications for glioma therapy in humans remain unclear, we observed a significant improvement in animal survival upon down-regulation of MMP14 in murine intracranial glioma xenografts infected with MMP14 shRNA-expressing CRAd. We further found that down-regulation of MMP14 in gliomas by combinational treatment with CRAd-S-5/3 and Marimastat, a chemical inhibitor of metalloproteinases, augments suppression of pro-angiogenic factors, caused by the replication-competent adenovirus. We also demonstrated that delivery of MMP14-targeting shRNA by a fiber-modified adenoviral vector to the glioma cells effectively suppresses their proliferation in vitro and in vivo. Thus our data indicate that inhibition of MMP14 expression in tumors in combination with glioma virotherapy could be effectively utilized to suppress angiogenesis and neovascularization of glioma tumors by decreasing production of pro-angiogenic factors.

Tamoxifen improves cytopathic effect of oncolytic adenovirus in primary glioblastoma cells mediated through autophagy.

Oncolytic gene therapy using viral vectors may provide an attractive therapeutic option for malignant gliomas. These viral vectors are designed in a way to selectively target tumor cells and spare healthy cells. To determine the translational impact, it is imperative to assess the factors that interfere with the anti-glioma effects of the oncolytic adenoviral vectors. In the current study, we evaluated the efficacy of survivin-driven oncolytic adenoviruses pseudotyping with adenoviral fiber knob belonging to the adenoviral serotype 3, 11 and 35 in their ability to kill glioblastoma (GBM) cells selectively without affecting normal cells. Our results indicate that all recombinant vectors used in the study can effectively target GBM in vitro with high specificity, especially the 3 knob-modified vector. Using intracranial U87 and U251 GBM xenograft models we have also demonstrated that treatment with Conditionally Replicative Adenovirus (CRAd-S-5/3) vectors can effectively regress tumor. However, in several patient-derived GBM cell lines, cells exhibited resistance to the CRAd infection as evident from the diminishing effects of autophagy. To improve therapeutic response, tumor cells were pretreated with tamoxifen. Our preliminary data suggest that tamoxifen sensitizes glioblastoma cells towards oncolytic treatment with CRAd-S-5/3, which may prove useful for GBM in future experimental therapy.

Inhibition of MMP14 potentiates the therapeutic effect of temozolomide and radiation in gliomas.

Metalloproteinases are membrane-bound proteins that play a role in the cellular responses to antiglioma therapy. Previously, it has been shown that treatment of glioma cells with temozolomide (TMZ) and radiation (XRT) induces the expression of metalloproteinase 14 (MMP14). To investigate the role of MMP14 in gliomagenesis, we used several chemical inhibitors which affect MMP14 expression. Of all the inhibitors tested, we found that Marimastat not only inhibits the expression of MMP14 in U87 and U251 glioma cells, but also induces cell cycle arrest. To determine the relationship between MMP14 inhibition and alteration of the cell cycle, we used an RNAi technique. Genetic knockdown of MMP14 in U87 and U251 glioma cells induced G2/M arrest and decreased proliferation. Mechanistically, we show that TMZ and XRT regulated expression of MMP14 in clinical samples and in vitro models through downregulation of microRNA374. In vivo genetic knockdown of MMP14 significantly decreased tumor growth of glioma xenografts and improved survival of glioma-bearing mice. Moreover, the combination of MMP14 silencing with TMZ and XRT significantly improved the survival of glioma-bearing mice compared to a single modality treatment group. Therefore, we show that the inhibition of MMP14 sensitizes tumor cells to TMZ and XRT and could be used as a future strategy for antiglioma therapy.

The involvement of Notch signaling in melanoma vasculogenic mimicry.

Notch signaling plays an important role in tumor angiogenesis. Recent studies suggest that Notch signaling also regulates the progression of primary melanomas toward an aggressive phenotype. The aim of this study was to investigate the involvement of Notch signaling pathway in organization of tumor cells into capillary-like structures (CLS), the phenomenon also known as vasculogenic mimicry (VM). Here, we show that Notch signaling cascade was constitutively active in melanoma cell lines we used. Blocking Notch signaling with the γ-secretase inhibitors, DAPT, dibenzazepine or Jagged1 neutralizing antibody resulted in stabilization of CLS indicating that Notch signaling pathway attenuates melanoma VM. We further studied this phenomenon on melanomas grafted in nude mice. Compared to control, VM channels in DAPT-treated grafted melanoma became larger and more branched. DAPT-treated melanomas also exhibited an up-regulation of MMP-2 and VEGFR1, both known as VM mediators. Moreover, we did not observe necrosis in VM channels areas of DAPT-treated melanomas. These findings indicate that VM regulated by Notch signaling may present a novel target in melanoma therapy.

Three-dimensional in vivo imaging of tumors expressing red fluorescent proteins.

3D imaging of genetically-engineered fluorescent tumors enables quantitative monitoring of tumor growth/regression, metastatic processes, including during anticancer therapy in real-time.Fluorescent tumor models for 3D imaging require stable expression of genetically encoded fluorescent proteins and maintenance of the properties of tumor cell line including growth rate, morphology, and immunophenotype.In this chapter, the protocol for 3D imaging of tumors expressing red fluorescent protein are described in detail.

Clinical significance of KISS1 protein expression for brain invasion and metastasis.

BACKGROUND: Metastases to the brain represent a feared complication and contribute to the morbidity and mortality of breast cancer. Despite improvements in therapy, prognostic factors for development of metastases are lacking. KISS1 is a metastasis suppressor that demonstrates inhibition of metastases formation in several types of cancer. The purpose of this study was to determine the importance of KISS1 expression in breast cancer progression and the development of intracerebral lesions. METHODS: In this study, we performed a comparative analysis of 47 brain metastases and 165 primary breast cancer specimens by using the antihuman KISS1 antibody. To compare KISS1 expression between different groups, we used a 3-tier score and the automated score computer software (ACIS) evaluation. To reveal association between mRNA and protein expression, we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Significance of immunohistochemistry stainings was correlated with clinicopathological data. RESULTS: We identified that KISS1 expression is significantly higher in primary breast cancer compared with brain metastases (P < .05). The mRNA analysis performed on 33 selected ductal carcinoma brain metastatic lesions and 36 primary ductal carcinomas revealed a statistically significant down-regulation of KISS1 protein in metastatic cases (P = .04). Finally, we observed a significant correlation between expression of KISS1 and metastasis-free survival (P = .04) along with progression of breast cancer and expression of KISS1 in primary breast cancer specimens (P = .044). CONCLUSIONS: In conclusion, our study shows that breast cancer expresses KISS1. Cytoplasmic expression of KISS1 may be used as a prognostic marker for increased risk of breast cancer progression.

VEGFR1 and PKCα signaling control melanoma vasculogenic mimicry in a VEGFR2 kinase-independent manner.

We have recently shown that vascular endothelial growth factor-A (VEGFA), a major regulator of tumor vascularization, is essential for the organization of tumor cells into capillary-like structure (CLS), which is a hallmark of tumor vasculogenic mimicry (VM). Herein we further dissect the involvement of VEGFA and its downstream transducers, VEGF receptor 1 (VEGFR1), VEGFR2, and protein kinase C (PKC) in melanoma VM. The knockdown of VEGFR1 in three melanoma cell lines completely disrupts Matrigel-induced CLS formation, whereas inhibition of VEGFR2 kinase with a specific inhibitor, protein tyrosine kinase inhibitor II (PTKi-II), does not affect the process, indicating that VEGFR2 signaling is not involved in VEGFA-mediated melanoma VM. Furthermore, among tested PKC isoforms, only PKCα and δ are expressed in the melanoma cells during CLS formation. Pretreatment with selective PKCα and δ inhibitors blocked CLS formation. However, inhibition of PKCα, but not PKCδ, completely destroyed the previously formed CLS. Moreover, knockdown of PKCα, but not PKCδ, using small interfering RNAs abrogated CLS formation, suggesting that PKCα is the major contributory factor in melanoma VM. In-vivo experiments indicate that disruption of PKCα signaling significantly reduces the signs of VM in allografted B16/F10 melanoma. These findings may contribute to the development of new therapeutic agents that target melanoma VM.

Melanoma vasculogenic mimicry capillary-like structure formation depends on integrin and calcium signaling.

OBJECTIVE: We recently demonstrated that the formation of CLSs in vitro, which are thought to be a reconstitution of VM, is controlled by VEGFA. CLS formation also requires the extracellular matrix signals, presumably transduced by integrins. Both pathways are affected by Ca(2+). Therefore, we directly tested the roles of Ca(2+) and integrin in melanoma VM. METHODS: The investigation was performed by immunocytochemical, histochemical, and 3D co-culture assays. We have also used an in vivo animal model. RESULTS: The extracellular and intracellular Ca(2+) chelators, EGTA and BAPTA-AM, prevented CLS formation on Matrigel, caused actin rearrangement, and completely destroyed the preformed CLS. Addition of colcemid or cytochalasin D prevented the CLS formation and destroyed the preformed CLS network. Herein, we also show that blocking antibodies to ανß3 and ανß5 integrins disrupted the CLS network. Control blocking antibody to ß1 integrin had no effect. In vivo experiments indicated that Ca(2+) chelation dramatically reduced the signs of VM in melanoma tumors grafted in mice. CONCLUSIONS: Our results indicate that the formation of CLS is tightly regulated by extracellular and intracellular Ca(2+) levels; ανß3 and ανß5 integrins are primarily responsible for CLS formation, whereas ß1 integrin does not participate in CLS formation.

Prognostic significance of periodic acid-Schiff-positive patterns in clear cell renal cell carcinoma.

BACKGROUND: The ability of aggressive tumors to form nonendothelial tumor cell-lined microvascular channels is known as "vasculogenic mimicry" (VM). VM channels are revealed as periodic acid-Schiff (PAS)-positive patterns, and in some tumors their presence predicts clinical outcomes. OBJECTIVE: We aimed to study VM channels in clear cell renal cell carcinoma (cRCC) tumors and explore their prognostic significance and relationship to other suggested prognostic factors such as thymidine phosphorylase (TP) and vascular endothelial growth factor (VEGF) expression. METHODS: We retrospectively studied 45 patients who had undergone radical nephrectomy for clinically confined cRCC (stage T2-T3NOMO) at the Russian Cancer Research Center. The tumor sections were reviewed for disease stage, nuclear grade, perirenal fat invasion, and lymph node involvement, and we performed immunohistochemical staining for VEGF and TP expression, and PAS staining. Disease-free survival probabilities were determined by Kaplan-Meier estimates and prognostic factors were evaluated by univariate analysis. RESULTS: PAS-positive patterns observed in the cRCC tumor included back-to-back closed loops, networks, arcs, and parallel patterns. There was a significant decrease in disease-free survival among patients with PAS-positive networks (p = 0.005), but not among patients with other PAS-positive patterns. TP expression was also a significant predictor of disease-free survival (p = 0.035), but this factor did not correlate with the presence of PAS-positive networks. Notably, in our small sample, the six patients whose tumors were positive for both factors had the highest risk of cancer recurrence. CONCLUSIONS: The presence of PAS-positive networks is an independent and relevant prognostic parameter for disease-free survival in patients with cRCC. Our data suggest that the combination of PAS-positive networks and TP expression may identify patients with the highest risk of cancer recurrence.

Cancer/testis genes expression in human melanoma cell lines.

We analyzed the expression of 15 cancer/testis and four melanoma differentiation antigens in 21 metastatic melanoma cell lines using reverse transcriptase-polymerase chain reaction (RT-PCR) assay. On the basis of morphological characteristics, tumor cell lines were divided into three groups with high, moderate, and low grade of differentiation. Evaluation of gene expression and melanoma cell morphology has revealed a correlation between increased expression of cancer/testis genes and differentiation grade of cancer cells. The gene expression pattern for lymph node metastases and primary tumors exhibits the distribution of expression level and frequency similar to that found for established cell lines. Nevertheless, only 60% lymph node metastases or primary tumor tissue of randomly selected patients show marked expression of the most prominent cancer/testis genes, and almost 90% lesion tissue expresses at least one of 15 cancer/testis genes.

Melanoma vasculogenic mimicry is strongly related to reactive oxygen species level.

The concept of 'vasculogenic mimicry' (VM) was introduced to describe the unique ability of highly invasive tumor cells to form capillary-like structures (CLS) and matrix-rich patterned network in three-dimensional culture that mimic embryonic vasculogenic network. Recently, we have shown that CLS formation requires apoptotic cell death through activation of caspase-3-dependent mechanism. In this study, to identify some molecular determinants driving aggressive melanoma cells to express a latent 'angiogenic program' that recapitulates the early events of CLS formation, we focused on the involvement of antioxidants (AOs) in the process of melanoma VM. We have studied the effects of resveratrol, (-)-epigallocathechin gallate, N-acetyl-cysteine (NAC) and Trolox on the ability of melanoma cells to form/destroy CLS. We observed that the formation of CLS was strongly related to reactive oxygen species level. In vivo animal experiments confirmed the involvement of reactive oxygen species level in melanoma VM. To understand the molecular mechanisms of this phenomenon, we specifically looked for induction of apoptosis and vascular endothelial growth factor (VEGF) release. Western blot analysis revealed that the level of VEGF, VEGF receptors (VEGF-Rs) and active caspase-3 dramatically decreased in cells treated with AOs. Here, we also report further experiments designed to determine whether the crosstalk between AOs and apoptosis exists in melanoma VM.

Crosstalk between apoptosis and antioxidants in melanoma vasculogenic mimicry.

The concept of "vasculogenic mimicry" (VM) was introduced to describe the unique ability of highly aggressive tumor cells to form capillary-like structure (CLS) and matrix-rich patterned network in three-dimensional cultures that mimic embryonic vasculogenic network. Here, we provide the experimental evidence that CLS structure formation requires apoptotic cell death through activation of caspase-dependent mechanism. Our results indicate that the formation of CLS is also related to the reactive oxygen species (ROS) levels.

Serum levels of soluble HLA and IL-2R molecules in patients with urogenital chlamydia infection.

Cellular immunity plays a central role in immune response to chlamydial infection, and soluble forms of immune cell membrane antigens take part in the regulation of immune response. Using an immunoenzymatic method, we determined serum levels of soluble HLA molecules (sHLA-I and sHLA-DR) and soluble CD25 molecules (sCD25) in patients with genital chlamydial infection. Specimens from patients with nonspecific inflammation of the urogenital tract were studied and healthy volunteers served as controls. We revealed that serum levels of sHLA-DR and sCD25 increased 3.5- and 2.3-fold, respectively, during chlamydial infection, while the levels of sHLA-I were not changed. Nonspecific inflammation of the urogenital tract was characterized by a 1.5-fold increase in sHLA-I, a 1.6-fold decrease in sCD25, and no changes of sHLA-DR levels in comparison with healthy volunteers. We concluded that Th1 immune responses might dominate during genital chlamydial infection contrary to the state of nonspecific inflammation of urogenital tract.

Atemonate and Imuteran: novel Russian monoclonal antibody-based therapeutic agents.

The N. Blokhin National Cancer Research Center is one of the few Russian scientific institutions in which hybridoma technology of monoclonal antibody (mAb) production has been successfully established. Using this technology, several dozens of mAbs to various antigens of human leukocytes have been elaborated. These mAbs are widely used for immune status evaluation and for differential diagnostics of leukemias. Two mAbs were used to develop therapeutic drugs. Imuteran is a pharmaceutical form of mAb ICO-25 against a mucin-like antigen of human milk fat globules and proposed for treatment of epithelial cell-originating cancers (breast, intestinal, ovarian, lung cancer, etc.). ThePhase II clinical study of this agent is now nearly completed, and preliminary results suggest Imuteran to be a promising anticancer agent with tumor-stabilizing activity, but patients should be carefully monitored for signs of allergic reactions. mAb ICO-90 against the CD3 antigen of human T lymphocytes was used to develop the therapeutic agent Atemonate proposed for treatment of acute transplant rejection. At present, the Phase II clinical study of this agent is over, and the results confirm the drug safety and efficacy for this indication. The drug is being registered at the Ministry of Healthcare and Social Development, and transfer to serial production is expected shortly.

The involvement of apoptosis in melanoma vasculogenic mimicry.

During development, the formation and remodeling of the primary vascular network occurs by vasculogenesis and angiogenesis. In 1999, the concept of vasculogenic mimicry was introduced to describe the unique ability of highly aggressive tumor cells to form a capillary-like structure and a matrix-rich patterned network in three-dimensional culture that mimic the embryonic vasculogenic network. In this study, we examined the ability of melanoma cells derived from patients with disseminated melanoma to engage in vasculogenic mimicry in order to identify key parameters in the complexity of the formation of capillary-like structure. We showed that disseminated melanoma as well as uveal and cutaneous melanoma adopts a vascular-related phenotype and engages in vasculogenic mimicry: the main geometrical features of capillary-like structure are determined during the first step of the vascular network assembly. We provided experimental evidence that capillary-like structure formation requires apoptotic cell death through activation of a caspase-dependent mechanism: a broad range caspase inhibitor zVAD-fmk and a caspase-3 inhibitor DEVD blocked capillary-like structure formation. Apoptosis occurs before capillary-like structure formation but not after capillary-like structures have assembled. These observations may provide a better understanding of the mechanisms involved in melanoma vasculogenic mimicry.

Effect of brain injury on immunophenotype of peripheral blood lymphocytes in rats.

Strong immunosuppression occurs after severe traumatic brain injury (TBI) and most likely contributes substantially to the patient morbidity and mortality. However, the mechanisms of this immunosuppression are unknown. For the lowering of stressful factors, severe TBI was induced in anaesthetized rats. The lymphocyte subsets from 60 rats with severe TBI were analyzed using monoclonal antibody by the indirect immunofluorescence method. The blood of 30 rats without TBI was used as a control. When compared to the control group, the rats with TBI showed a remarkable reduction in the relative number of CD4(+), RT-Ia(+), Thy-1(+) and ICO-111(+) lymphocytes during the first 2 h after injury. Further reduction in the number of CD4(+) cells was determined in the rats during all the period of the experimental observation. The number of lymphocytes expressing membrane Thy-1 and ICO-111 antigens was significantly decreased 7 days after the trauma. The relative number of RT-Ia(+) lymphocytes was significantly reduced in rats with TBI during 14 days following the trauma. A significant decrease in the luminescence intensity of all the analyzed antigen-positive cells was also observed in rats with TBI. Between the 7th and the 14th days after the trauma a positive correlation between the number of Thy-1(+) PBLs and the number of RT-Ia(+) lymphocytes was determined. Similar results on lymphocyte immunophenotype were seen in patients with TBI. Thus, the cellular immune response is identical in patients and in animals with TBI. Severe brain traumatic injury leads to a reduced expression of cell surface antigens and causes a decrease in the number of antigen-positive lymphocytes and in the intensity of their luminescence.

Immunophenotype of Peripheral Blood Lymphocytes in Patients with Burns.

Severe immunosuppression occurs after large thermal burns and probably contributes substantially to patient morbidity and mortality. The mechanism of immunosuppression is much unknown. T lymphocyte subsets from 45 severely patients with burns and 35 healthy donors were analyzed using monoclonal antibodies by indirect immunofluorescence method. Compared to healthy donors, patients with burns have shown a profound reduction in relative number of CD3(+) lymphocytes during the 24-h period following injury, which was accompanied by a decrease in CD4 but not CD8 subsets. Activated lymphocytes, while determined by the expression of CD25, CD26 and CD71, were insignificantly increased in patients with burns. The expression HLA DR was insignificantly decreased on peripheral blood lymphocytes from thermally injured patients. Additionally, the significant decrease of CD95 antigen expression was observed in all patients with burns. Also, significant decrease of the luminescence intensity in all analyzed antigens was observed in patients with burns. Numerous positive correlations between CD3(+), CD8(+), CD25(+), CD26(+) and CD71(+) cells were revealed, especially during the first week after thermal trauma. We conclude that the thermal injury produces a profound relative CD3(+) and CD4(+) cell lymphopenia with signs of moderate lymphocyte activation.

Influence of Immune Complexes on Expression of CD54 and CD95 on the Surface of Endothelial Cells of ECV-304 Line.

The etiology of rheumatoid arthritis (RA) remains unknown; however, recent studies have suggested a central role of the vascular endothelium in RA pathogenesis. The immune complex (IC)-mediated vasculitis is typical for RA. The studies reported herein were undertaken to determine 1) whether IC isolated from plasma of patients with RA are capable of inducing expression of ICAM-1/CD54 and Fas antigen/CD95 on the endothelial cell (EC) in vitro, 2) whether the capacity to induce expression of this phenotypic markers of EC activation is determined by the size or by the composition of the IC. The concentrations of IC were chosen to be within the range for IC levels in plasma. We have shown that all IC-containing samples (n = 8) significantly and in a dose-dependent manner increased level of ICAM-1 expression on the EC. In selected experiments EC were incubated with IC in the presence of complement. The presence of serum containing active complement components resulted in further up-regulation of ICAM-1 expression only in 4 of 8 cases, independently on the ability of IC to fix complement. Additionally, we have found that all IC samples significantly and in a dose-dependent manner induced the marked increase in CD95 expression on the EC. Furthermore, the levels of augmented expression of CD95 correlated with the levels of CD54 expression stimulated by the same IC-containing samples. We have demonstrated that these levels of stimulated expression of ICAM-1 as well as levels of Fas antigen expression negatively correlated with percentage amounts of IgM in total protein concentration of IC and directly correlated with IgG content in comparison to IgM in the structure of this IC. Our results show that IC stimulate ECs to express ICAM-1 and CD95, all of which have been implicated in the pathogenesis of rheumatic disease. The studies reported herein provide further information regarding to inflammatory potential of IC in RA.