Placenta growth factor induces melanoma resistance to temozolomide through a mechanism that involves the activation of the transcription factor NF-κB

Placenta growth factor (PlGF) and its receptor vascular endothelial growth factor receptor-1 (VEGFR-1) are co-expressed in a large number of human melanoma cell lines. Moreover, a correlation between in vivo PlGF production and melanoma progression has been suggested. To investigate whether PlGF might have a role in protecting melanoma cells from the cytotoxic effects of the anticancer agent temozolomide (TMZ), which is used for the treatment of this malignancy, we stably transfected a doxycycline-inducible PlGF antisense mRNA into a human melanoma cell clone that secretes VEGF-A and PlGF and expresses receptors for both growth factors. Induction of PlGF antisense mRNA in the transfected cells (13443/ASP3 subclone) halved TMZ IC(50), and exogenous addition of PlGF to the culture medium 24 h before TMZ treatment, partially restored IC(50) values to that of control cells. The increased sensitivity of 13443/ASP3 cells upon PlGF antisense mRNA expression was not due to down-regulation of O6-methylguanine-DNA methyltransferase, a DNA repair protein that represents the main mechanism of resistance to TMZ. Since the activity of the transcription factor nuclear factor-κB (NF-κB) has been correlated to melanoma chemoresistance, we investigated whether NF-κB was involved in PlGF-induced melanoma cell resistance to TMZ. Induction of PlGF antisense mRNA in 13443/ASP3 cells halved the levels of active NF-κB and the specific inhibition of this transcription factor increased sensitivity of 13443/ASP3 cells to TMZ. In conclusion, our data strongly suggest that PlGF plays a role in melanoma cell resistance to TMZ through a pathway that involves NF-κB activation

Antitumor activity of a novel anti-vascular endothelial growth factor receptor-1 monoclonal antibody that does not interfere with ligand binding

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase transmembrane receptor that has also a soluble isoform containing most of the extracellular ligand binding domain (sVEGFR-1). VEGF-A binds to both VEGFR-2 and VEGFR-1, whereas placenta growth factor (PlGF) interacts exclusively with VEGFR-1. In this study we generated an anti-VEGFR-1 mAb (D16F7) by immunizing BALB/C mice with a peptide that we had previously reported to inhibit angiogenesis and endothelial cell migration induced by PlGF. D16F7 did not affect binding of VEGF-A or PlGF to VEGFR-1, thus allowing sVEGFR-1 to act as decoy receptor for these growth factors, but it hampered receptor homodimerization and activation. D16F7 inhibited both the chemotactic response of human endothelial, myelomonocytic and melanoma cells to VEGFR-1 ligands and vasculogenic mimicry by tumor cells. Moreover, D16F7 exerted in vivo antiangiogenic effects in a matrigel plug assay. Importantly, D16F7 inhibited tumor growth and was well tolerated by B6D2F1 mice injected with syngeneic B16F10 melanoma cells. The antitumor effect was associated with melanoma cell apoptosis, vascular abnormalities and decrease of both monocyte/macrophage infiltration and myeloid progenitor mobilization. For all the above, D16F7 may be exploited in the therapy of metastatic melanoma and other tumors or pathological conditions involving VEGFR-1 activation

Ellagic acid inhibits bladder cancer invasiveness and in vivo tumor growth

Ellagic acid (EA) is a polyphenolic compound that can be found as a naturally occurring hydrolysis product of ellagitannins in pomegranates, berries, grapes, green tea and nuts. Previous studies have reported the antitumor properties of EA mainly using in vitro models. No data are available about EA influence on bladder cancer cell invasion of the extracellular matrix triggered by vascular endothelial growth factor-A (VEGF-A), an angiogenic factor associated with disease progression and recurrence, and tumor growth in vivo. In this study, we have investigated EA activity against four different human bladder cancer cell lines (i.e., T24, UM-UC-3, 5637 and HT-1376) by in vitro proliferation tests (measuring metabolic and foci forming activity), invasion and chemotactic assays in response to VEGF-A and in vivo preclinical models in nude mice. Results indicate that EA exerts anti-proliferative effects as a single agent and enhances the antitumor activity of mitomycin C, which is commonly used for the treatment of bladder cancer. EA also inhibits tumor invasion and chemotaxis, specifically induced by VEGF-A, and reduces VEGFR-2 expression. Moreover, EA down-regulates the expression of programmed cell death ligand 1 (PD-L1), an immune checkpoint involved in immune escape. EA in vitro activity was confirmed by the results of in vivo studies showing a significant reduction of the growth rate, infiltrative behavior and tumor-associated angiogenesis of human bladder cancer xenografts. In conclusion, these results suggest that EA may have a potential role as an adjunct therapy for bladder cancer

Temozolomide and cisplatin in relapsed/refractory acute leukemia

Cisplatin depletes MGMT and increases the sensitivity of leukemia cells to temozolomide. We performed a phase I study of cisplatin and temozolomide in patients with relapsed and refractory acute leukemia. Fifteen patients had AML, 3 had ALL, and 2 had biphenotypic leukemia. The median number of prior chemotherapy regimens was 3 (1–5). Treatment was well tolerated up to the maximal doses of temozolomide 200 mg/m2/d times 7 days and cisplatin 100 mg/m2 on day 1. There was one complete remission in this heavily pretreated patient population. Five of 20 (25%) patients demonstrated a significant reduction in bone marrow blasts

EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

EMMPRIN's expression in melanoma tissue was reported to be predictive of poor prognosis. Here we demonstrate that EMMPRIN up-regulated VEGF receptor-2 (VEGFR-2) in two different primary melanoma cell lines and consequently increased migration and proliferation of these cells while inhibiting their apoptosis. SiRNA inhibition of VEGFR-2 expression abrogated these EMMPRIN effects. EMMPRIN regulation of VEGFR-2 was mediated through the over-expression of HIF-2α and its translocation to the nucleus where it forms heterodimers with HIF-1β. These results were supported by an in vivo correlation between the expression of EMMPRIN with that of VEGFR-2 in human melanoma tissues as well as with the extent of HIF-2α localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2α/VEGFR-2 mediated mechanism, with an autocrine role in melanoma cell malignancy. The inhibition of EMMPRIN in cancer may thus simultaneously target both the VEGFR-2/VEGF system and the matrix degrading proteases to block tumor cell growth and invasion

Endothelin-1 Inhibits Prolyl Hydroxylase Domain 2 to Activate Hypoxia-Inducible Factor-1α in Melanoma Cells

The endothelin B receptor (ET(B)R) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1alpha is essential for melanomagenesis and progression, and is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) and subsequent proteosomal degradation.Here we found that in melanoma cells ET-1, ET-2, and ET-3 through ET(B)R, enhance the expression and activity of HIF-1alpha and HIF-2alpha that in turn regulate the expression of vascular endothelial growth factor (VEGF) in response to ETs or hypoxia. Under normoxic conditions, ET-1 controls HIF-alpha stability by inhibiting its degradation, as determined by impaired degradation of a reporter gene containing the HIF-1alpha oxygen-dependent degradation domain encompassing the PHD-targeted prolines. In particular, ETs through ET(B)R markedly decrease PHD2 mRNA and protein levels and promoter activity. In addition, activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ET(B)R-mediated PHD2 inhibition, HIF-1alpha, HIF-2alpha, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced in vitro tube formation of endothelial cells and melanoma cell invasiveness, demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanoma tissues as well as in cell lines, that express high levels of HIF-1alpha, ET(B)R expression is associated with low PHD2 levels. In melanoma xenografts, ET(B)R blockade by ET(B)R antagonist results in a concomitant reduction of tumor growth, angiogenesis, HIF-1alpha, and HIF-2alpha expression, and an increase in PHD2 levels.In this study we identified the underlying mechanism by which ET-1, through the regulation of PHD2, controls HIF-1alpha stability and thereby regulates angiogenesis and melanoma cell invasion. These results further indicate that targeting ET(B)R may represent a potential therapeutic treatment of melanoma by impairing HIF-1alpha stability

Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Therapeutic implication of vascular endothelial growth factor receptor-1 (VEGFR-1) targeting in cancer cells and tumor microenvironment by competitive and non-competitive inhibitors

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor for VEGF-A, VEGF-B, and placental growth factor (PlGF) ligands that is expressed in endothelial, myelomonocytic and tumor cells. VEGF-B and PlGF exclusively bind to VEGFR-1, whereas VEGF-A also binds to VEGFR-2. At variance with VEGFR-2, VEGFR-1 does not play a relevant role in physiological angiogenesis in the adult, while it is important in tumor-associated angiogenesis. VEGFR-1 and PlGF are expressed in a variety of tumors, promote invasiveness and contribute to resistance to anti-VEGF-A therapy. The currently approved antiangiogenic therapies for the treatment of a variety of solid tumors hamper VEGF-A signaling mediated by both VEGFR-2 and VEGFR-1 [i.e., the monoclonal antibody (mAb) anti-VEGF-A bevacizumab, the chimeric molecule aflibercept and several small molecule tyrosine kinase inhibitors] or exclusively by VEGFR-2 (i.e., the mAb anti-VEGFR-2 ramucirumab). However, molecules that interfere with VEGF-A/VEGFR-2 signaling determine severe adverse effects due to inhibition of physiological angiogenesis and their efficacy is hampered by tumor infiltration of protumoral myeloid cells. Blockade of VEGFR-1 may exert anti-tumor activity by multiple mechanisms: a) inhibition of tumor-associated angiogenesis; b) reduction of myeloid progenitor mobilization and tumor infiltration by VEGFR-1 expressing M2 macrophages, which contribute to tumor progression and spreading; c) inhibition of invasiveness, vasculogenic mimicry and survival of VEGFR-1 positive tumor cells. As a consequence of these properties, molecules targeting VEGFR-1 are expected to produce less adverse effects and to counteract resistance towards anti-VEGF-A therapies. More interestingly, selective VEGFR-1 inhibition might enhance the efficacy of immunotherapy with immune checkpoint inhibitors. In this review, we will examine the experimental evidence available so far that supports targeting VEGFR-1 signal transduction pathway for cancer treatment by competitive inhibitors that prevent growth factor interaction with the receptor and non-competitive inhibitors that hamper receptor activation without affecting ligand binding