microRNA-130a suppresses breast cancer cell migration and invasion by targeting FOSL1 and upregulating ZO-1.

FOSL1 is frequently overexpressed in multiple types of human cancers including invasive breast cancers and implicated in cancer invasion and metastasis. However, how FOSL1 is overexpressed in cancers remains to be elucidated. Several microRNAs (miRNAs) have been shown to target FOSL1 and are downregulated in human cancers. Here, we report that miR-130a is a novel FOSL1 targeting miRNA. Using gene expression microarray analysis, we found that FOSL1 is among the most up-regulated genes in cells transfected with miR-130a inhibitors. Transient transfection-immunoblot, RNA-immunoprecipitation, and luciferase reporter assays revealed that miR-130a directly targets FOSL1 mRNA at its 3'-UTR. Overexpression of miR-130a significantly reduced the levels of FOSL1 in invasive breast cancer MDA-MB-231 and Hs578T cell lines and suppresses their migration and invasion. This inhibition can be rescued by ectopic expression of miR-130a-resistant FOSL1. Interestingly, we show that overexpression of miR-130a increased the levels of tight-junction protein ZO-1 while inhibition of miR-130a reduced the levels of ZO-1. We further show that miR-130a expression is significantly reduced in cancer tissues from triple-negative breast cancer (TNBC) patients, correlating significantly with the upregulation of FOSL1 expression, compared to non-TNBC tissues. Together, our results reveal that miR-130a directly targets FOSL1 and suppresses the inhibition of ZO-1, thus inhibiting cancer cell migration and invasion, in TNBCs.

Functional regulation of hypoxia inducible factor-1α by SET9 lysine methyltransferase.

HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells.

HIF1α protein stability is increased by acetylation at lysine 709.

Lysine acetylation regulates protein stability and function. p300 is a component of the HIF-1 transcriptional complex and positively regulates the transactivation of HIF-1. Here, we show a novel molecular mechanism by which p300 facilitates HIF-1 activity. p300 increases HIF-1α (HIF1α) protein acetylation and stability. The regulation can be opposed by HDAC1, but not by HDAC3, and is abrogated by disrupting HIF1α-p300 interaction. Mechanistically, p300 specifically acetylates HIF1α at Lys-709, which increases the protein stability and decreases polyubiquitination in both normoxia and hypoxia. Compared with the wild-type protein, a HIF1α K709A mutant protein is more stable, less polyubiquitinated, and less dependent on p300. Overexpression of the HIF1α wild-type or K709A mutant in cancer cells lacking the endogenous HIF1α shows that the K709A mutant is transcriptionally more active toward the HIF-1 reporter and some endogenous target genes. Cancer cells containing the K709A mutant are less sensitive to hypoxia-induced growth arrest than the cells containing the HIF1α wild-type. Taken together, these data demonstrate a novel biological consequence upon HIF1α-p300 interaction, in which HIF1α can be stabilized by p300 via Lys-709 acetylation.

Malate dehydrogenase 2 confers docetaxel resistance via regulations of JNK signaling and oxidative metabolism.

BACKGROUND: Resistance to chemotherapy represents a significant obstacle in prostate cancer therapeutics. Novel mechanistic understandings in cancer cell chemotherapeutic sensitivity and resistance can optimize treatment and improve patient outcome. Molecular alterations in the metabolic pathways are associated with cancer development; however, the role of these alterations in chemotherapy efficacy is largely unknown. METHODS: In a bed-side to bench-side reverse translational approach, we used cDNA microarray and qRT-PCR to identify genes that are associated with biochemical relapse after chemotherapy. Further, we tested the function of these genes in cell proliferation, metabolism, and chemosensitivity in prostate cancer cell lines. RESULTS: We report that the gene encoding mitochondrial malate dehydrogenase 2 (MDH2) is overexpressed in clinical prostate cancer specimens. Patients with MDH2 overexpression had a significantly shorter period of relapse-free survival (RFS) after undergoing neoadjuvant chemotherapy. To understand the molecular mechanism underlying this clinical observation, we observed that MDH2 expression was elevated in prostate cancer cell lines compared to benign prostate epithelial cells. Stable knockdown of MDH2 via shRNA in prostate cancer cell lines decreased cell proliferation and increased docetaxel sensitivity. Further, MDH2 shRNA enhanced docetaxel-induced activations of JNK signaling and induced metabolic inefficiency. CONCLUSION: Taken together, these data suggest a novel function for MDH2 in prostate cancer development and chemotherapy resistance, in which MDH2 regulates chemotherapy-induced signal transduction and oxidative metabolism.

HDAC4 protein regulates HIF1α protein lysine acetylation and cancer cell response to hypoxia.

Hypoxia-inducible factor 1 α (HIF1α) is an essential part of the HIF-1 transcriptional complex that regulates angiogenesis, cellular metabolism, and cancer development. In von Hippel-Lindau (VHL)-null kidney cancer cell lines, we reported previously that HIF1α proteins can be acetylated and inhibited by histone deacetylase (HDAC) inhibitors or specific siRNA against HDAC4. To investigate the mechanism and biological consequence of the inhibition, we have generated stable HDAC4 knockdown via shRNA in VHL-positive normal and cancer cell lines. We report that HDAC4 regulates HIF1α protein acetylation and stability. Specifically, the HIF1α protein acetylation can be increased by HDAC4 shRNA and decreased by HDAC4 overexpression. HDAC4 shRNA inhibits HIF1α protein stability. In contrast, HDAC1 or HDAC3 shRNA has no such inhibitory effect. Mutations of the first five lysine residues (lysine 10, 11, 12, 19, and 21) to arginine within the HIF1α N terminus reduce protein acetylation but render the mutant HIF1α protein resistant to HDAC4 and HDACi-mediated inhibition. Functionally, in VHL-positive cancer cell lines, stable inhibition of HDAC4 decreases both the HIF-1 transcriptional activity and a subset of HIF-1 hypoxia target gene expression. On the cellular level, HDAC4 inhibition reduces the hypoxia-related increase of glycolysis and resistance to docetaxel chemotherapy. Taken together, the novel biological relationship between HDAC4 and HIF1α presented here suggests a potential role for the deacetylase enzyme in regulating HIF-1 cancer cell response to hypoxia and presents a more specific molecular target of inhibition.

Anthracycline chemotherapy inhibits HIF-1 transcriptional activity and tumor-induced mobilization of circulating angiogenic cells.

Using a cell-based reporter gene assay, we screened a library of drugs in clinical use and identified the anthracycline chemotherapeutic agents doxorubicin and daunorubicin as potent inhibitors of hypoxia-inducible factor 1 (HIF-1)-mediated gene transcription. These drugs inhibited HIF-1 by blocking its binding to DNA. Daily administration of doxorubicin or daunorubicin potently inhibited the transcription of a HIF-1-dependent reporter gene as well as endogenous HIF-1 target genes encoding vascular endothelial growth factor, stromal-derived factor 1, and stem cell factor in tumor xenografts. CXCR4(+)/sca1(+), VEGFR2(+)/CD34(+), and VEGFR2(+)/CD117(+) bone-marrow derived cells were increased in the peripheral blood of SCID mice bearing prostate cancer xenografts but not in tumor-bearing mice treated for 5 days with doxorubicin or daunorubicin, which dramatically reduced tumor vascularization. These results provide a molecular basis for the antiangiogenic effect of anthracycline therapy and have important implications for refining the use of these drugs to treat human cancer more effectively.

Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization.

HIF-1 is a heterodimeric transcription factor that mediates adaptive responses to hypoxia and plays critical roles in cancer progression. Using a cell-based screening assay we have identified acriflavine as a drug that binds directly to HIF-1alpha and HIF-2alpha and inhibits HIF-1 dimerization and transcriptional activity. Pretreatment of mice bearing prostate cancer xenografts with acriflavine prevented tumor growth and treatment of mice bearing established tumors resulted in growth arrest. Acriflavine treatment inhibited intratumoral expression of angiogenic cytokines, mobilization of angiogenic cells into peripheral blood, and tumor vascularization. These results provide proof of principle that small molecules can inhibit dimerization of HIF-1 and have potent inhibitory effects on tumor growth and vascularization.

Identifying phenotype-associated subpopulations by integrating bulk and single-cell sequencing data.

Single-cell RNA sequencing (scRNA-seq) distinguishes cell types, states and lineages within the context of heterogeneous tissues. However, current single-cell data cannot directly link cell clusters with specific phenotypes. Here we present Scissor, a method that identifies cell subpopulations from single-cell data that are associated with a given phenotype. Scissor integrates phenotype-associated bulk expression data and single-cell data by first quantifying the similarity between each single cell and each bulk sample. It then optimizes a regression model on the correlation matrix with the sample phenotype to identify relevant subpopulations. Applied to a lung cancer scRNA-seq dataset, Scissor identified subsets of cells associated with worse survival and with TP53 mutations. In melanoma, Scissor discerned a T cell subpopulation with low PDCD1/CTLA4 and high TCF7 expression associated with an immunotherapy response. Beyond cancer, Scissor was effective in interpreting facioscapulohumeral muscular dystrophy and Alzheimer's disease datasets. Scissor identifies biologically and clinically relevant cell subpopulations from single-cell assays by leveraging phenotype and bulk-omics datasets.

Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth.

A library of drugs that are in clinical trials or use was screened for inhibitors of hypoxia-inducible factor 1 (HIF-1). Twenty drugs inhibited HIF-1-dependent gene transcription by >88% at a concentration of 0.4 microM. Eleven of these drugs were cardiac glycosides, including digoxin, ouabain, and proscillaridin A, which inhibited HIF-1alpha protein synthesis and expression of HIF-1 target genes in cancer cells. Digoxin administration increased latency and decreased growth of tumor xenografts, whereas treatment of established tumors resulted in growth arrest within one week. Enforced expression of HIF-1alpha by transfection was not inhibited by digoxin, and xenografts derived from these cells were resistant to the anti-tumor effects of digoxin, demonstrating that HIF-1 is a critical target of digoxin for cancer therapy.

HIF1 and ID1 Interplay Confers Adaptive Survival to HIF1α-Inhibition.

Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.

Supraphysiologic Testosterone Induces Ferroptosis and Activates Immune Pathways through Nucleophagy in Prostate Cancer.

The discovery that androgens play an important role in the progression of prostate cancer led to the development of androgen deprivation therapy (ADT) as a first line of treatment. However, paradoxical growth inhibition has been observed in a subset of prostate cancer upon administration of supraphysiologic levels of testosterone (SupraT), both experimentally and clinically. Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive prostate cancer cells. This was initiated by the induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activated nucleic acid sensors converge on NFκB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT resulted in increased migration of cytotoxic immune cells to tumor beds in xenograft models and patient tumors. Collectively, these findings indicate that SupraT may inhibit a subset of prostate cancer by activating nucleic acid sensors and downstream immune signaling. SIGNIFICANCE: This study demonstrates that supraphysiologic testosterone induces two parallel autophagy-mediated processes, ferritinophagy and nucleophagy, which then activate nucleic acid sensors to drive immune signaling pathways in prostate cancer.

CCL2 is induced by chemotherapy and protects prostate cancer cells from docetaxel-induced cytotoxicity.

BACKGROUND: Metastatic prostate cancer is either inherently resistant to chemotherapy or rapidly acquires this phenotype after chemotherapy exposure. In this study, we identified a docetaxel-induced resistance mechanism centered on CCL2. METHODS: We compared the gene expression profiles in individual human prostate cancer specimens before and after exposure to chemotherapy collected from previously untreated patients who participated in a clinical trial of preoperative chemotherapy. Subsequently, we used the gain- and loss-of-function approach in vitro to identify a potential mechanism underlying chemotherapy resistance. RESULTS: Among the molecular signatures associated with treatment, several genes that regulate the inflammatory response and chemokine activity were upregulated including a significant increase in transcripts encoding the CC chemokine CCL2. Docetaxel increased CCL2 expression in prostate cancer cell lines in vitro. CCL2-specific siRNA inhibited LNCaP and LAPC4 cell proliferation and enhanced the growth inhibitory effect of low-dose docetaxel. In contrast, overexpression of CCL2 or recombinant CCL2 protein stimulated prostate cancer cell proliferation and rescued cells from docetaxel-induced cytotoxicity. This protective effect of CCL2 was associated with activation of the ERK/MAP kinase and PI3K/AKT, inhibition of docetaxel-induced Bcl2 phosphorylation at serine 70, phosphorylation of Bad, and activation of caspase-3. The addition of a PI3K/AKT inhibitor Ly294002 reversed the CCL2 protection and was additive to docetaxel-induced toxicity. CONCLUSION: These results support a mechanism of chemotherapy resistance mediated by cellular stress responses involving the induction of CCL2 expression and suggest that inhibiting CCL2 activity could enhance therapeutic responses to taxane-based therapy.

Prostate cancer-associated gene expression alterations determined from needle biopsies.

PURPOSE: To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. EXPERIMENTAL DESIGN: Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays composed of 6,200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative reverse transcription-PCR. RESULTS: Comparative analyses identified 954 transcript alterations associated with cancer (q < 0.01%), including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy use, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of androgen receptor expression changes was noted. In exploratory analyses, androgen receptor down-regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. CONCLUSIONS: Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.

Molecular Crosstalk Between MYC and HIF in Cancer.

The transcription factor c-MYC (MYC thereafter) is a global regulator of gene expression. It is overexpressed or deregulated in human cancers of diverse origins and plays a key role in the development of cancers. Hypoxia-inducible factors (HIFs), a central regulator for cells to adapt to low cellular oxygen levels, is also often overexpressed and activated in many human cancers. HIF mediates the primary transcriptional response of a wide range of genes in response to hypoxia. Earlier studies focused on the inhibition of MYC by HIF during hypoxia, when MYC is expressed at physiological level, to help cells survive under low oxygen conditions. Emerging evidence suggests that MYC and HIF also cooperate to promote cancer cell growth and progression. This review will summarize the current understanding of the complex molecular interplay between MYC and HIF.

The iroquois homeobox gene 5 is regulated by 1,25-dihydroxyvitamin D3 in human prostate cancer and regulates apoptosis and the cell cycle in LNCaP prostate cancer cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most active metabolite of vitamin D3, has significant antitumor activity in a broad range of preclinical models of cancer. In this study, we show that the Iroquois homeobox gene 5 (Irx5) is down-regulated by 1,25(OH)2D3 in human prostate cancer samples from patients randomly assigned to receive weekly high-dose 1,25(OH)2D3 or placebo before radical prostatectomy. Down-regulation of Irx5 by 1,25(OH)2D3 was also shown in the human androgen-sensitive prostate cancer cell line LNCaP and in estrogen-sensitive MCF-7 breast cancer cells. Knockdown of Irx5 by RNA interference showed a significant reduction in LNCaP cell viability, which was accompanied by an increase in p21 protein expression, G2-M arrest, and an increase in apoptosis. The induced apoptosis was partially mediated by p53, and p53 protein expression was increased as a result of Irx5 knockdown. Cell survival was similarly reduced by Irx5 knockdown in the colon cancer cell line HCT 116 and in MCF-7 breast cancer cells, each being derived from clinical tumor types that seem to be inhibited by 1,25(OH)2D3. Overexpression of Irx5 led to a reduction of p21 and p53 expression. This is the first report that Irx5 is regulated by 1,25(OH)2D3 in humans and the first report to show that Irx5 is involved in the regulation of both the cell cycle and apoptosis in human prostate cancer cells. Irx5 may be a promising new therapeutic target in cancer treatment.

Combination strategy targeting the hypoxia inducible factor-1 alpha with mammalian target of rapamycin and histone deacetylase inhibitors.

PURPOSE: The hypoxia-inducible factor-1 alpha (HIF-alpha) is a key regulator of tumor angiogenesis. Mammalian target of rapamycin (mTOR) and histone deacetylase (HDAC) inhibitors suppress tumor-induced angiogenesis by reducing tumor HIF-1 alpha protein expression. Thus, we hypothesized that combination treatment of rapamycin and the HDAC inhibitor LBH589 has greater antiangiogenic and antitumor activity compared with single agents. EXPERIMENTAL DESIGN: To evaluate the effect of LBH589 and rapamycin on HIF-1 alpha in human prostate PC3, renal C2 carcinoma cell lines, and endothelial cells (human umbilical vein endothelial cells), we did Western blot analysis. To determine the antitumor activity of LBH589 and rapamycin, cell proliferation assays and xenograft experiments were conducted. RESULTS: Western blotting showed that combination treatment of human umbilical vein endothelial cells, C2 and PC3, significantly reduced HIF-1 alpha protein expression compared with single agents. Treatment with rapamycin resulted in inhibition of the downstream signals of the mTOR pathway and increased phosphorylation of Akt in C2 cells, whereas the constitutively activated Akt in PC3 cells was not modulated. LBH589 decreased both constitutively expressed and rapamycin-induced phosphorylated Akt levels in PC3 and C2 cell lines. In clonogenic assays, the combination treatment had a greater inhibitory effect in PC3 cells (93 +/- 1.4%) compared with single agents (66 +/- 9% rapamycin and 43 +/- 4% LBH589). Combination of rapamycin and LBH589 significantly inhibited PC3 and C2 in vivo tumor growth and angiogenesis as measured by tumor weight and microvessel density. CONCLUSIONS: Combination treatment of mTOR and HDAC inhibitors represents a rational therapeutic strategy targeting HIF-1 alpha that warrants clinical testing.

Publisher Correction: Interplay between hypoxia and androgen controls a metabolic switch conferring resistance to androgen/AR-targeted therapy.

The original version of this Article contained errors in Fig. 7. In panels e and f, the graph titles incorrectly read 'LNCaP-AdtNs' and 'LAPC4-AdtNs', respectively. These errors have now been corrected in both the PDF and HTML versions of the Article.

Vascular endothelial growth factor trap blocks tumor growth, metastasis formation, and vascular leakage in an orthotopic murine renal cell cancer model.

PURPOSE: Angiogenesis inhibitors have shown clinical benefit in patients with advanced renal cell cancer, but further therapeutic improvement is needed. Vascular endothelial growth factor (VEGF) Trap is a newly developed VEGF-blocking agent with stronger affinity and broader activity than the anti-VEGF antibody bevacizumab. In this study, we tested the activity of VEGF Trap in an orthotopic murine model of renal cancer with spontaneous lung metastases. EXPERIMENTAL DESIGN: Murine syngeneic renal cell carcinoma cells (RENCA) transfected with a luciferase-expressing vector were injected into the renal capsule of BALB/c mice. I.p. treatment with VEGF Trap or control protein (10 or 25 mg/kg twice weekly) was started shortly after tumor injection to prevent tumor development (prevention model) or after established tumors were formed to inhibit tumor growth and metastasis formation (intervention model). RESULTS: In the prevention model, VEGF Trap inhibited tumor growth by 87 +/- 14% compared with control (P=0.007) and significantly prolonged survival. In the intervention model, VEGF Trap inhibited tumor growth by 74 +/- 9% (P<0.001) and the formation of lung metastases was inhibited by 98% (P<0.004). Microvascular density was reduced by 66% due to VEGF Trap treatment (P<0.001). In addition, VEGF Trap prevented fibrinogen leakage into the tumor microenvironment representative for reduced vascular leaking as shown by immunohistochemical staining. CONCLUSIONS: VEGF Trap is a potent inhibitor of RENCA tumor growth and metastasis formation and blocks the biological function of VEGF in vivo. These results support further clinical development of VEGF Trap for renal cell cancer and other cancer types.

Platelets take up the monoclonal antibody bevacizumab.

PURPOSE: One of the key factors that promotes angiogenesis is vascular endothelial growth factor (VEGF). Platelets are the main source of VEGF in blood and contribute to angiogenesis by release of growth factors, including VEGF, from their alpha-granules on activation. The monoclonal antibody bevacizumab blocks VEGF in the blood of patients within hours after administration. Platelets are known to endocytose plasma proteins including immunoglobulins. We tested the hypothesis that platelets take up bevacizumab. EXPERIMENTAL DESIGN: Fluorescence-activated cell sorting analysis, immunofluorescence imaging, and Western blotting were used to study uptake and release of bevacizumab by platelets in vitro and in vivo. The angiogenic activity of platelets preincubated with bevacizumab was studied in endothelial proliferation assays. Finally, we determined whether treatment with bevacizumab neutralizes VEGF in platelets from cancer patients. RESULTS: We found that platelets are able to take up bevacizumab. Activation of platelets preincubated with bevacizumab resulted in release of the antibody and release of VEGF neutralized by bevacizumab. Immunofluorescence microscopy revealed that FITC-labeled bevacizumab and P-selectin colocalize, indicating alpha-granule localization. In addition, bevacizumab uptake inhibited platelet-induced human endothelial cell proliferation. In in vivo rabbit experiments, FITC-labeled bevacizumab was present in platelets after 2 h and up to 2 weeks following i.v. administration. Finally, we found that platelets take up bevacizumab in patients receiving bevacizumab treatment. Within 8 h after bevacizumab administration, platelet VEGF was almost completely neutralized due to this uptake. CONCLUSION: These studies show that bevacizumab is taken up by platelets and may explain its clinical effect on wound healing and tumor growth.

Antitumor effect of the histone deacetylase inhibitor LAQ824 in combination with 13-cis-retinoic acid in human malignant melanoma.

Resistance to chemotherapy is a major hurdle in the treatment of malignant melanoma. Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in different tumor types, including melanoma, and to reverse epigenetic repression of tumor suppressor genes, such as retinoic acid receptor beta (RARbeta). In this study, we tested the antitumor effect of the HDAC inhibitor LAQ824 in combination with 13-cis-retinoic acid (CRA) on two human melanoma cell lines both in vitro and in vivo. Treatment of LAQ824 showed a dose-dependent inhibitory effect on A2058 and HMV-I cell lines in a clonogenic assay. These cell lines were relatively resistance to CRA. On treatment with combination of LAQ824 and CRA, a greater inhibitory effect (up to 98%) was achieved compared with single agents. Lack of RARbeta2 gene expression was associated with histone acetylation and gene methylation at the promoter level. Treatment with LAQ824 restored retinoid sensitivity by reverting RARbeta2 epigenetic silencing. The biological effect of LAQ824 was associated with p21 induction in both cell lines but G(2) cell cycle arrest in A2058 and apoptosis in HMV-I cell line. The induction of apoptosis by LAQ824 was associated with increased reactive oxygen species and induction of SM22 gene expression in HMV-I but not in A2058 cell line. Administration of the free radical scavenger l-N-acetylcysteine blocked LAQ824 + CRA-mediated apoptosis in HMV-I cells, suggesting a primary role for reactive oxygen species generation in LAQ824 + CRA-associated lethality. Combination treatment showed 61% and 82% growth inhibition in A2058 and HMV-I tumors, respectively. Greater induction of in vivo apoptosis was observed in the HMV-I but not in the A2058 tumors treated with combination therapy compared with single agents. These results suggest that the HDAC inhibitor LAQ824 has a greater antitumor activity in combination with CRA in melanoma tumors but the degree of induced apoptosis may vary. Combination of HDAC inhibitors and retinoids represents a novel therapeutic approach for malignant melanoma that warrants clinical testing.