HIF-1alpha Expression Research Articles

ALPPS procedure in an experimental colorectal liver metastases model: hero or villain?

Background: Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been raised as a potential therapy for bilobar metastatic liver disease, although the potential tumor progression (TP) that this technique may induce is still not well established. Methods: The aim of this report was to study tumor hepatic progression induced by the first step of ALPPS in a WAG/Rij rat syngenic model of metastatic colorectal carcinoma by subcapsular inoculation of CC531 cell line. We analyzed the effect of hypoxia and immune response induced by ALPPS on liver metastases. Results: ALLPS seems to induce not only tumor progression, but also metastases independently of the site of inoculation. Immunohistochemical analisys revealed that ALPPS induced expression of hepatic vasculogenic factors and a dramatic increase of intrahepatic macrophages in comparison with non-operated groups. Interestingly, hepatic macrophages strongly expressed COX-2, while tumor-infiltrating macrophages expressed mainly arginase-1. ALPPS also induced a decrease of tumor-infiltrating lymphocytes and increase of intrahepatic T-lymphocytes. Conclusion: ALPPS technique may induce hypoxic environment which enhance hepatic HIF1alpha and VEGF expression. Additionally, the regenerative stimulus seems to be driven by a pro-inflammatory environment, in which M1 intrahepatic macrophages, which expressed COX-2 may develop a key role. These facts may be related with the tumor progression observed in inoculated and operated animals.

Disulfram/Copper Complex May Enhance Its Cytotoxic Effect on CD34+CD38- KG1-Alpha Cells By Down Regulating the Expression of HIF1-Alpha in the Co-Cultured MSCs

Backgroud We had reported that Disulfram/copper complex (DS/ Cu) had a potent and selective anti-leukemia property in vitro against leukemia stem-like cells (e.g., CD34+/CD38- KG1α and Kasumi-1 cells and primary CD34+ cells isolated from AML patients) as well as was highly effective in vivo in CD34+/CD38- leukemic cell-derived xenograft mouse models. Here, we report the in vitro activity of DS/Cu against CD34+/CD38- leukemia stem-like cells sorted from KG1α AML cell line by affecting the co-cultured AML bone marrow mesenchymal stromal cells. Methods KG1α cells were cultured and stained with hCD34-APC, hCD38- PE, and hCD123-PE-Cy7 to determine the percentage of cells expressing CD34, CD38, and CD123. To isolate CD38- cells, CD38 microbeads were used for the depletion of CD38+ cells. First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8) and monitor expression of HIF-1α expression by western blot. Next, the sorted CD34+CD38- KG1α cells were co-cultured with primary AML bone marrow MSCs isolated from a de novo AML patient in the hypoxic condition induced by Cobalt Chloride (CoCl2) for 24 hr. The co-cultured cells were treated with DS/CU with/without YC1(HIF-1α inhibitor)、IDA(idarubicin) and then separated. CCK8 assay, fow cytometry were used to determine the cell proliferation and apoptosis, respectively. Finally, we used western bolting to explore the underlying mechanism of the cytotoxicity of DS/CU in the AML cells and the sensitizated effect in AML MSCs. Results First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8). As shown in Figure 1, DS/Cu (DS 1umol/L,Cu 0.5umol/l) administrated alone was unable to induce apoptosis in MSCs (P>0.05 vs untreated control; see below Figure 1). However, DS in combination with 0.5 μM Cu for 24 hrs resulted in signifcantly increased apoptosis in CD34+CD38- KG1α cells.( P<0.001 vs untreated control; see Figure 1) .The co-cultured cells were treated with DS/CU、IDA with/without YC1 and then separated. CCK8 assay, fow cytometry were used to determine the cell apoptosis, respectively. Treatment with DS/CU resulted in signifcantly increased apoptosis in the co-cultured CD34+/CD38- KG1α cells (Figure 2; P<0.05, DS/Cu vs either untreated control). Comparable phenomena were observed in co-cultured CD34+/CD38- KG1α cells treated with idarubicin. However, while treatment with YC1 and DS/CU had no signifcant effect on cell apoptosis (P>0.05 vs untreated control). Taken together, these results indicated that DS/CU was active against co-cultured CD34+/CD38- KG1α cells and the effect can be reversed by HIF1-α inhibitor(see Figure 2).We examined the cytotoxic effect of DS/Cu on CD34+/CD38- KG1α cells in different culture systems using the CCK-8. As shown in Figure 4, while treatment with DS/CU had significant inhibited proliferation on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P<0.05 vs un-co-cultured control). However, while treatment with DS/CU and CoCl2 (HIF-1α promoter) had no signifcant effect on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P>0.05 vs un-co-cultured control,see Figure 3).CD34+CD38- KG1α cells and MSCs separated from the co-cultured system were treated with DS/CU with/without YC1 and Cocl2, followed by Western blot analysis to monitor expression of HIF-1α. As shown at the figure 4, the expressions of HIF-1α and SDF-1α between the two cells were clearly down-regulated treated with DS/CU alone. And the expressions of HIF-1α and SDF-1α were up-regulated treated with DS/CU and CoCl2. However, when co-treated with YC1, the down-regulation of HIF-1α was reverse. DS/Cu down redulated the expressions of HIF1-α、SDF1-α、CXCR4 and VLA4 in the co-cultured CD34+CD38- KG1α cells and these effect could be reserved by HIF1-α inhibitor.Conclusion DS/Cu preferentially induces apoptosis of CD34+CD38- KG1α cells, but not MSCs. DS/Cu exhibited cytotoxicity in CD34+/CD38- KG1α cells, and MSCs enhances this effect. The mechanism may be related to the HIF1α/SDF1/CXCR4 and VLA4 pathway. DS/CU may enhance its inhibitory effect on CD34+CD38- KG1α cells by down regulating the expression of HIF1-α in MSCS. DisclosuresNo relevant conflicts of interest to declare.

Associations between whole tumor histogram analysis parameters derived from ADC maps and expression of EGFR, VEGF, Hif 1-alpha, Her-2 and Histone 3 in uterine cervical cancer

ObjectiveDiffusion weighted imaging (DWI) can be quantified by apparent diffusion coefficient (ADC) and can predict tissue microstructure. The aim of the present study was to analyze possible associations between ADC histogram based parameters with different histopathological parameters in cervical squamous cell carcinoma. Materials and methods18 female patients (age range 32–79 years) with squamous cell cervical carcinoma were retrospectively enrolled. In all cases, pelvic MRI was performed with a DWI (b-values 0 and 1000 s/mm2). Histogram analysis was performed as a whole lesion measurement. Histopathological parameters included expression of EGFR, VEGF, Hif1-alpha, Her2 and Histone 3. Spearman's correlation coefficient was used to analyze associations between investigated parameters. ResultsAnalyze of the investigated ADC histogram parameters showed a good interreader variability, ranging from 0.705 for entropy to 0.959 for ADCmedian. EGFR expression correlated statistically significant with several histogram parameters. The highest correlation was observed for p75 (p = −0.562, P = 0.015). There were several correlations with histone 3, the highest with p25 (p = −0.610, P = 0.007). None of the ADC related parameters correlated statistically significant with expression of VEGF, Hif1-alpha and Her2. ConclusionHistogram analysis showed a good interreader agreement. ADC histogram parameters might be able to reflect expression of EGFR and histone 3 in cervical squamous cell carcinomas, but not expression of VEGF, Hif1-alpha and Her2.

Abstract B06: Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia

Abstract Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved over the past few decades, approximately 30% of patients relapse. The overall survival (OS) rate of pediatric patients with primary refractory or relapsed AML is about 30%. Resistance to therapy presents a great challenge for AML patients. Therefore, there is a critical need to understand mechanisms of resistance, as well as to identify novel therapies that will improve OS. The leukemia-initiating cells reside in special hypoxic niches in the bone marrow. Thus, studying drug response of AML cells within a hypoxic environment is essential for more accurate prediction of patients’ response to therapy. We performed functional drug screening using AML cell lines, which demonstrated a differential response of AML cell lines to the survivin-targeting agent YM155 in normoxia versus hypoxia. Survivin (BIRC5), an inhibitor of apoptosis, is overexpressed in a variety of pediatric blood cancers compared to normal cells. We hypothesize that survivin may be an attractive target for therapy in pediatric AML. The overall objective of this study was to study mechanisms of AML response to YM155 in normoxia and hypoxia and survivin’s potential as a therapeutic target in pediatric AML. Purpose: The aims of the present work were (1) to study BIRC5 expression in publicly available databases of pediatric AML including the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, and (2) to determine the potential mechanisms of hypoxia-altered response to YM155 in AML cells. Methods and Results: In the present study, the TARGET AML gene-expression profiling data available on Affymetrix human gene 1.1 ST arrays (HuGene 1.1) were used to study BIRC5 expression in comparison to HuGene 1.1 datasets from Gene Expression Omnibus (GEO) containing profiling data from adult AML and normal tissues. BIRC5 expression was significantly higher in pediatric AML in comparison to cytogenetically normal (CN) adult AML, and in relapsed pediatric AML in comparison to matched samples at diagnosis. In order to identify novel targeted therapies in AML we used a panel of AML cell lines to screen a library of 50 compounds in normoxia and hypoxia. The survivin-targeting agent YM155 was found to be one of the most cytotoxic compounds in the majority of AML cell lines in normoxia. However, hypoxia conferred resistance to this drug. The IC50 of YM155 in AML cell lines in normoxia ranged from 1.5 nM-8 µM, with an increase of 2-100 fold in hypoxia (IC50 from 22 nM-14 µM). The Mv4-11 cell line that harbors a FLT3-ITD mutation was selected for further mechanistic studies. Hypoxia conferred resistance to YM155 in Mv4-11 cells through inhibition of apoptosis. In order to identify the underlying molecular mechanisms, we performed RNA-seq of YM155-treated Mv4-11 cells in normoxia and hypoxia, and identified a set of over-represented genes that were validated by quantitative RT-PCR. The mRNA expression of HIF2alpha, p21WAF1, and STAT3 was overexpressed after 9h of YM155 treatment, while PIM2 was downregulated in normoxia but not in hypoxia, in comparison to vehicle-treated control. Preliminary data suggest a role of stem cell genes in hypoxia-induced resistance to YM155. Ongoing studies are directed towards determining the functions of the identified genes in resistance. Conclusion: Our data demonstrate that survivin (BIRC5) is overexpressed in pediatric AML in comparison to adult CN-AML, and may be an attractive target for pediatric AML therapy. Additionally, YM155 is highly cytotoxic to AML cell lines in normoxia; however, for its effective use in the clinic, cotargeting hypoxia-induced resistance factors is essential. Citation Format: David W. Lee, Justin Montoya, Kieran Finch, Jack Wilkinson-Dix, Robert J. Arceci, Daniel H. Wai, Eiman Aleem. Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B06.

ADC-histogram analysis in head and neck squamous cell carcinoma. Associations with different histopathological features including expression of EGFR, VEGF, HIF-1α, Her 2 and p53. A preliminary study

ObjectiveApparent diffusion coefficient (ADC) values derived from Diffusion-weighted images are able to reflect tumor microstructure, such as cellularity, extracellular matrix or proliferation potential. This present study sought to correlate prognostic relevant histopathologic parameters with ADC values derived from a whole lesion measurement in head and neck squamous cell carcinoma (HNSCC). Materials and methodsThirty-four patients with histological proven primary HNSCC were prospectively acquired. Histogram analysis was derived from ADC maps. In all cases, expression of Hif1-alpha, VEGF, EGFR, p53, p16, Her 2 were analyzed. ResultsIn the overall patient sample, ADCmax correlated with p53 expression (p = −0.446, p = 0.009) and ADCmode correlated with Her2-expression (p = −0.354, p = 0.047). In the p16 positive group there were several correlations. P25, P90 and entropy correlated with Hif1-alpha (p = −0.423, p = 0.05, p = −0.494, p = 0.019, p = 0.479, p = 0.024, respectively). Kurtosis correlated with P53 expression (p = −0.466, p = 0.029).For p16 negative carcinomas the following associations could be identified. Mode correlated with VEGF-expression (p = −0.657, p = 0.039). ADCmax, P75, P90, and Std correlated with p53-expression (p = −0.827, p = 0.002, p = −0.736, p = 0.01, p = −0.836, p = 0.001 and p = −0.70, p = 0.016, respectively). There were no statistically significant differences of ADC histogram parameters between p16 positive and p16 negative carcinomas. ConclusionADC histogram values can reflect different histopathological features in HNSCC. Associations between ADC histogram analysis parameters and histopathology depend on p16 status.

FA05.05: PROLONGED LAPAROSCOPIC GASTRIC ISCHEMIC CONDITIONING PRIOR TO ESOPHAGECTOMY IN PATIENTS OF ESOPHAGEAL CANCER: FEASIBILITY AND CLINICAL SIGNIFICANCE

Abstract Background The studies on gastric ischemic preconditioning in improving blood flow to the distal gastric conduit and reducing the incidence of anastomotic complications have yielded variable results. Lack of adequate time interval between gastric ischemic preconditioning and esophagectomy has been postulated as one of the reasons for lack of clinical benefit. Hence the present study was done to evaluate the feasibility and clinical outcome of prolonged gastric ischemic conditioning prior to esophagectomy. Methods Patients with locally advanced squamous cell carcinoma of the esophagus planned for neoadjuvant chemoradiotherapy followed by esophagectomy and gastric pull through were included in the prospective study. Prior to initiation of neoadjuvant therapy laparoscopic feeding jejunostomy with ischemic preconditioning by the ligation of left and the short gastric vessel was performed. All patients underwent thoracolaparoscopic esophagectomy with two field esophagectomy and cervical esophagogastric anastomosis using modified Orringer technique. Radiological assessment of the caliber of the right gastroepiploic artery (RGEA) and Immunohistologic analysis of Hypoxia-inducible factor – 1 alpha (HIF-1alpha) expression in the fundus to determine the degree of neovascularization was performed after ischemic preconditioning. The feasibility of laparoscopic gastric mobilization post ischemic preconditioning and cervical anastomotic leak rate was documented and compared with patients who did not undergo ischemic preconditioning. Results Fifteen patients underwent thoracolaparoscopic esophagectomy after a median time of 124 days post laparoscopic gastric ischemic preconditioning. Minor adhesions present in the gastrosplenic region did not interfere with laparoscopic gastric mobilization. The mean pre-ischemic conditioning diameter of RGEA increased from 2.18 mm to 2.25 mm post ischemic conditioning (P = 0.21). The median HIF-1 alpha index post ischemic conditioning was 0.6. The cervical anastomotic leak rate was less in patients who underwent ischemic preconditioning (1/14, 7.1%) compared to the patients who did not undergo ischemic preconditioning (6/16, 37.5%), although the difference was not statistically significant (P = 0.24) Conclusion Prolonged gastric ischemic preconditioning prior to esophagectomy is safe and does not interfere with laparoscopic gastric mobilization. It can potentially reduce cervical anastomotic complications post neoadjuvant chemoradiotherapy. Disclosure All authors have declared no conflicts of interest.

Abstract A28: Beta-escin inhibits ovarian cancer metastasis by targeting the tumor microenvironment

Abstract The high mortality rate that results from ovarian cancer (OvCa) is caused by the wide dissemination of cancer cells within the abdominal cavity. OvCa cells metastasize to the peritoneum, which is covered by a single layer of mesothelial cells, and invade into the underlying stroma, composed of extracellular matrices including fibronectin (FN) and stromal cells such as fibroblasts. Using a three-dimensional organotypic quantitative high-throughput screening platform (3D-qHTS) modeling the peritoneum, we discovered that beta-escin inhibited OvCa adhesion and invasion to the peritoneum. Beta-escin is a natural triterpenoid saponin from Chinese horse chestnut seeds and has a variety of known pharmacologic effects. We hypothesize that beta-escin targets both the cancer cells and the stromal cells to inhibit the metastatic niche. The therapeutic effects of beta-escin and horse chestnut seed extract were tested in vivo using xenograft and syngeneic models of OvCa prevention and intervention. The mechanisms of beta-escin action in cancer cells and microenvironmental cells was explored in vitro using OvCa cell lines and primary human mesothelial cells and fibroblasts, and in vivo using different mouse models of metastasis. Moreover, a collection of 160 analogs of beta-escin were gathered and screened using our 3D-qHTS platform for inhibitory activity in OvCa cell adhesion/invasion. Our results reveal that beta-escin and horse chestnut extract taken orally inhibit metastasis in both OvCa prevention and intervention models. In addition, beta-escin mechanistically depresses the pluripotent stem cell population, inflammatory cytokine secretion, HIF-1alpha expression, and fibronectin production, while it increases autophagy in the tumor microenvironment. Furthermore, three distant analogs of beta-escin inhibited OvCa metastasis, and all three are cardiac glycosides. Taken together, we reveal a potential therapeutic value for beta-escin and/or analogs of beta-escin in preventing and treating OvCa dissemination. Citation Format: Hilary A. Kenny, Madhu Lal, Min Shen, Betul Kara, Chun-Yi Chiang, Karen Watters, Peter Hart, Marc Ferrer, Ernst Lengyel. Beta-escin inhibits ovarian cancer metastasis by targeting the tumor microenvironment. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A28.

Serum and Tissue HIF-2 Alpha Expression in CIN, N-Acetyl Cysteine, and Sildenafil-Treated Rat Models: An Experimental Study

Background and Objectives: Contrast-induced nephropathy (CIN), is acute renal damage due to contrast agents. This study is conducted to evaluate serum and renal heterodimeric nuclear transcription factor (HIF)-2 alpha levels and its tissue expression in contrast-induced nephropathy, and in N-acetyl cysteine (NAC)-and Sildenafil-treated rat models. Materials/Methods: This randomized, controlled, interventional animal study was conducted on Wistar rats. Rats (n = 36) were randomly assigned to four groups: control (n = 9), CIN group (n = 9), CIN + NAC group (n = 9), and sildenafil (n = 9). The rat model was used to form iohexol-originated CIN. During the modeling, prophylactic treatment was performed at the 24th and 48th h. After 48 h of modeling, blood, urine, and tissue samples were obtained for biochemical analyses. HIF-2-α levels were measured in renal tissue, serum, and urine samples. Renal sections were also performed for histopathologic and immunohistochemical evaluations of renal injury and HIF-2-α expression. Results: In the CIN model, HIF-2α levels and other biochemical parameters were significantly increased (p < 0.01). Both sildenafil and NAC efficiently decreased renal damage due to contrast agents, as shown in histopathologic examinations (p < 0.05). Similarly, after treatment with sildenafil and NAC, HIF-2α levels were significantly decreased (p < 0.05). Conclusions: The current study shows that serum and tissue HIF-2α levels decrease in CIN. Besides, the levels and tissue expression of HIF-2α decrease with both NAC and sildenafil treatments. With further studies, HIF-2α can be investigated as a biomarker of CIN and can be used in the follow-up of patients with CIN.

Increased HIF-1alpha And VEGF Expression Found in Various T Stages of Clear Cell Renal Cell Carcinoma

There is an increase in renal cell carcinoma in males aged over fifty years with smoking risk factors. Clear cell renal cell carcinoma is the most common malignant tumor found in the kidneys that have an aggressive nature. This carcinoma is classified according to TNM into T1, T2, T3 and T4 stages. The higher the T stage, the worse the prognosis of the patient, as it involves the role of the HIF-1a transcription factor accumulated under hypoxic condition due to inactivation of VHL, thus activating VEGF. So far, clear cell renal cell carcinoma frequently found in smoking males of fifth decade of age or older has not been explained.. An observational analytic study was conducted on the sample of paraffin blocks of patients with clear cell renal cell carcinoma in 2010-2016. T1, T2, T3, and T4 stages of 9 samples, 12 samples, 12 samples and 6 samples, respectively, were collected in Anatomic Pathology Laboratory, Dr. Soetomo Hospital, Surabaya. Detection of HIF-1a and VEGF expression was performed using immunohisto-chemical immunity with HIF-1a and VEGF antibodies. The results were analyzed statistically by Kruskal-Wallis and Mann-Whitney methods. The correlation between HIF-1a and VEGF in T stages was analyzed using Spearman method. There was a difference of HIF-1a expression between stage T1 with T2, T1 with T3, T1 with T4 (p=0.041, p=0.000, and p=0.002), stage T2 with T3 and stage T2 with T4 (p=0.001, and p=0.013). There was a difference in VEGF expression in stage T1 with T2, T1 with T3, T1 with T4 (p=0.007, p=0.000, and p=0.002), and stage T2 with T3 (p=0,001). There was a correlation between HIF-1a and VEGF expressions with all T stages (rs=0.624 with p 0.000). As a conclusion, the higher the stage of T, the higher the expression of HIF-1a and VEGF. The higher the levels of HIF-1a and VEGF, the worse the prognosis of clear cell renal carcinoma. HIF-1a and VEGF play a role in the pathogenesis of clear cell renal cell carcinoma. Thus, HIF-1a and VEGF can be developed as prognostic markers. Increased Hif-1aAnd Vegf Expression Found In Various T Stages Of Clear Cell Renal Cell Carcinoma

Weak HIF-1alpha expression indicates poor prognosis in resectable pancreatic ductal adenocarcinoma

BackgroundHIF-1alpha and CAIX proteins are commonly expressed under hypoxic conditions, but other regulatory factors have been described as well. Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia and strong stromal reaction and has a dismal prognosis with the currently available treatment modalities.MethodsWe investigated the expression and prognostic role of HIF-1alpha and CAIX in PDAC series from Northern Finland (n = 69) using immunohistochemistry.ResultsIn our PDAC cases, 95 and 85% showed HIF-1alpha and CAIX expression, respectively. Low HIF-1alpha expression correlated with poor prognosis, and multivariate analysis identified weak HIF-1alpha intensity as an independent prognostic factor for PDAC-specific deaths (HR 2.176, 95% CI 1.216–3.893; p = 0.009). There was no correlation between HIF-1alpha and CAIX expression levels, and the latter did not relate with survival.ConclusionsOur findings are in contrast with previous research by finding an association between low HIF-1alpha and poor prognosis. The biological mechanisms remain speculative, but such an unexpected relation with prognosis and absence of correlation between HIF-1alpha and CAIX suggests that the prognostic association of HIF-1alpha may not directly be linked with hypoxia. Accordingly, the role of HIF-1alpha might be more complex than previously thought and the use of this marker as a hypoxia-related prognostic factor should be addressed with caution.

DIPG-67. HYPOXIA-INDUCIBLE FACTORS REGULATE DIFFUSE INTRINSIC PONTINE GLIOMA GROWTH IN NORMOXIC CULTURE

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways.

Downregulation of HIF complex in the hypothalamus exacerbates diet-induced obesity

Hypothalamic hypoxia-inducible factor-1 (HIF-1) can regulate whole-body energy homeostasis in response to changes in blood glucose, suggesting that it acts as a sensor for systemic energy stores. Here, we hypothesized that hypothalamic HIF-1 could be affected by diet-induced obesity (DIO).We used eight-week old, male C57Bl6 mice, fed normal chow diet or with high fat diet for 1, 3, 7, 14 and 28 days. The expression of HIF-1alpha and HIF-1beta was measured by PCR and western blotting and its hypothalamic distribution was evaluated by fluorescence microscopy. Inhibition of HIF-1beta in arcuate nucleus of hypothalamus was performed using stereotaxic injection of shRNA lentiviral particles and animals were grouped under normal chow diet or high fat diet for 14 days.Using bioinformatics, we show that in humans, the levels of HIF-1 transcripts are directly correlated with those of hypothalamic transcripts for proteins involved in inflammation, regulation of apoptosis, autophagy, and the ubiquitin/proteasome system; furthermore, in rodents, hypothalamic HIF-1 expression is directly correlated with the phenotype of increased energy expenditure. In mice, DIO was accompanied by increased HIF-1 expression. The inhibition of hypothalamic HIF-1 by injection of an shRNA resulted in a further increase in body mass, a decreased basal metabolic rate, increased hypothalamic inflammation, and glucose intolerance.Thus, hypothalamic HIF-1 is increased during DIO, and its inhibition worsens the obesity-associated metabolic phenotype. Thus, hypothalamic HIF-1 emerges as a target for therapeutic intervention against obesity.

P-016 - Impact of HIF-1alpha and PKM1 expression on acquisition of paclitaxel resistance in gastric cancer

P-016 - Impact of HIF-1alpha and PKM1 expression on acquisition of paclitaxel resistance in gastric cancer

A phase I/II study of regorafenib (R) plus avelumab (A) in digestive tumors.

TPS3125 Background: Several preclinical studies have shown that simultaneous blockade of programmed death(PD) 1/PD-L1 and neoangiogenesis induces synergistic anti-tumour effect in vivo. R is a multikinase inhibitor approved for the treatment of metastatic colorectal (CRC) and gastro intestinal stromal tumor (GIST). A is a PD-L1 inhibitor approved for the treatment of Merkel carcinoma. We hypothesized that R in association with A could be synergistic and feasible in patients (pts) with tumors of the digestive tract. Methods: This is a multicenter, prospective phase I/II trial assessing R+A in 4 cohorts of pts with advanced pretreated: A) CRC not MSI-H or MMR-deficient, B) GIST, C) Oesophageal/gastric carcinoma, D) Biliary tract/hepatocellular carcinoma. Primary objectives are to determine the recommended phase II dose (RP2D) of R+A in phase I, and assess the best overall response defined as per RECIST v1.1 with A+R in phase II. In phase I, 2 doses of R will be investigated: 120mg, 160mg, daily, 3 weeks on/1 week off with fixed dose of A: 10mg/kg every 2 weeks. In phase II, all pts will received the RP2D of R with A. Main eligibility criteria are: -adult pts with metastatic, histologically confirmed tumor of 1 of the 4 cohorts, with measurable, progressive disease, after ≥ 1 previous line of systemic therapy. Primary endpoint is toxicity according to NCI-CTCAE v4.0 and incidence rate of DLT at each dose level during the first 28 days (I); antitumor activity in terms of best overall response (II). Secondary endpoints encompass: Objective Response Rate (ORR), Progression Free Survival (PFS), Growth modulation index, 6-months,1-year PFS and Overall Survival, PKs, Pharmacodynamics on (I) mandatory blood samples at baseline/on treatment, archived tumor tissue, (II) on optional biopsy at Baseline/after 4 weeks of R+A focusing on TAM, Lymphocytes infiltrates, PD-L1, VEGFR, PDGFR, HIF1alpha expression. Phase I will follow a classical 3+3 design with 2 dose levels and a maximum of 12 pts. A Bayesian approach will be used in phase II, with a maximum sample size of 50 pts/cohort. At each update of interim analysis, a stopping rule for inefficacy will recommend stopping the trial if there is a high predictive probability (≥80%) that ORR is < or = to the futility bound p0 = 20%. Clinical trial information: NCT03475953.

Abstract 504: MicroRNA Signatures in Murine Aorta and Carotid Arteries

Atherosclerosis, a major cause of mortality, affects arteries diffusely. However, some vascular beds are preferably involved. In the last decade microRNAs (miRs) have emerged as key regulators of gene expression in physiological or pathophysiological processes. Here, we investigated whether different arteries, commonly affected by atherosclerosis, present specific miR expression signatures. For this purpose, aorta (Ao) and carotid (Ca) arteries of four healthy male Wistar rats were dissected, total RNA was extracted with Trizol, and enriched for small RNA fraction. MicroRNAs were then sequenced using massive parallel sequencing (RNA-Seq) on Ion Torrent PGM platform. Data were analyzed using CLC Genomics Workbench software. We identified 266 mature miRs in Ao and 421 in Ca, 260 were common between arteries. Differential expression analysis (EDGE) showed increased expression (IE) of 16 miRs in Ao and reduced expression (RE) of 54 miRs in Ao, relative to Ca. The lists of differently expressed miRs were subjected to in silico functional analyzes: (1) computational target prediction, using miRWalk tool, identified 1,094 target genes for IE list and 3,574 for RE list; (2) gene-set enrichment analysis in the lists of putative target genes, using EnrichR tool, showed differences in enriched biological processes, such as response to shear stress, regulation of endothelial proliferation, smooth muscle metabolism, and response to FGF signaling (which were enriched in RE list); (3) construction of regulatory networks for differently expressed miRs, using miRnet, identified metabolic pathway components differently regulated between arteries, such as inhibition of SIRT1 and increased expression of HIF1-alpha in Ao. Taken together, our results show differences in miR signature between arteries, suggesting that specific expression profiles of miR may play a role in the vascular behavior as well as in atherosclerosis pathophysiology.

Histogram analysis of ADC in rectal cancer: associations with different histopathological findings including expression of EGFR, Hif1-alpha, VEGF, p53, PD1, and KI 67. A preliminary study

Functional imaging modalities like Diffusion-weighted imaging are increasingly used to predict tumor behavior like cellularity and vascularity in different tumors. Histogram analysis is an emergent imaging analysis, in which every voxel is used to obtain a histogram and therefore statistically information about tumors can be provided. The purpose of this study was to elucidate possible associations between ADC histogram parameters and several immunhistochemical features in rectal cancer. Overall, 11 patients with histologically proven rectal cancer were included into the study. There were 2 (18.18%) females and 9 males with a mean age of 67.1 years. KI 67-index, expression of p53, EGFR, VEGF, and Hif1-alpha were semiautomatically estimated. The tumors were divided into PD1-positive and PD1-negative lesions. ADC histogram analysis was performed as a whole lesion measurement using an in-house matlab application.Spearman's correlation analysis revealed a strong correlation between EGFR expression and ADCmax (p=0.72, P=0.02). None of the vascular parameters (VEGF, Hif1-alpha) correlated with ADC parameters. Kurtosis and skewness correlated inversely with p53 expression (p=-0.64, P=0.03 and p=-0.81, P=0.002, respectively). ADCmedian and ADCmode correlated with Ki67 (p=-0.62, P=0.04 and p=-0.65, P=0.03, respectively). PD1-positive tumors showed statistically significant lower ADCmax values in comparison to PD1-negative tumors, 1.93 ± 0.36 vs 2.32 ± 0.47×10−3mm2/s, p=0.04.Several associations were identified between histogram parameter derived from ADC maps and EGFR, KI 67 and p53 expression in rectal cancer. Furthermore, ADCmax was different between PD1 positive and PD1 negative tumors indicating an important role of ADC parameters for possible future treatment prediction.

The Role of HIF-1alpha in Regional Lymph Nodes Metastasis in Colorectal Adenocarcinoma

Colorectal carcinoma has high morbidity and mortality rate worldwide, in which over 90% are adenocarcinoma. Colorectal carcinoma is one of the most cancer that metastasize to the lymph node. Angiogenesis have an important role in tumor growth and metastasis. Hypoxia is the trigger factor for angiogenesis. Hypoxia induced factor-1 (HIF-1) is one critical protein directly reacting to hypoxia. HIF-1alpha, a HIF-1 subunit, is an important regulator of angiogenesis. This study analyzed HIF-1a expression in colorectal adenocarcinoma with and without regional lymph node metastasis. This study was to prove that HIF-1a has a role in regional lymph node metastasis in colorectal adenocarcinoma. An analytical observational study was conducted on thirty formalin fixed paraffin-embedded colorectal adenocarcinoma tissues from Anatomic Pathology Laboratory of Dr. Soetomo Hospital. Detection of HIF-1alpha expressions were performed with immunohistochemistry method, using HIF-1alpha antibody. It were statistically analyzed using Mann-Whitney and Kruskal- Wallis methods. There were no significant differences in the expression of HIF-1alpha in colorectal adenocarcinoma with or without lymph node (LN) metastasis (p&gt;0.05). As a conclusion, this study showed that HIF-1alpha has no role in LN metastasis in colorectal adenocarcinoma.

Characterizing The Effects of Ascorbic Acid and Hyperbaric Oxygen on Glioblastoma Cells

There is emerging evidence supporting the use of intravenous ascorbic acid (AA) as a pro‐oxidative, non‐toxic adjuvant therapy. Before AA is implicated in the clinic, however, more research needs to be conducted to better understand AA's mechanisms of action, and to determine what cancers would be most amenable to this adjunctive therapy. When administered intravenously, AA can achieve pharmacological, millimolar concentrations (~0.3–20 mM) and act as a pro‐oxidant in tumorous tissue, contrary to its antioxidant effects observed with oral administration, which produces micromolar, physiological concentrations (≤0.1 mM). Our study aims to evaluate the effects of pharmacological AA on glioblastoma cells (VM‐M3 and U87), and to see if another pro‐oxidative therapy, hyperbaric oxygen (HBOT), can sensitize cells to treatment. We hypothesize that 1‐hour treatment with AA will decrease VM‐M3 and U87 cell viability, and that co‐treatment with antioxidant N‐Acetylcysteine (NAC) will attenuate cell death. We also hypothesize that hydrogen peroxide mediates AA‐induced VM‐M3 and U87 cell death, and thus that exogenous catalase treatment will attenuate this effect. It was found that 1‐hour treatment with pharmacological AA decreased viability in both VM‐M3 and U87 cells, with VM‐M3 cells exhibiting greater sensitivity to treatment (assessed with crystal violet staining). HBOT did not enhance AA‐induced cytotoxicity in U87 cells; however, HBOT did sensitize VM‐M3 cells to AA treatment. Additionally, 1‐hour co‐treatment with AA and NAC, as well as AA and catalase, rescued cell death in U87 and VM‐M3 cells, suggesting that AA‐induced oxidative stress is mediated by hydrogen peroxide. This study also investigates the effects of AA and HBOT on HIF‐1 alpha, catalase, and transferrin receptor protein expression, yielding further insight into the pro‐oxidative role of AA in cancer.Support or Funding InformationWork supported by: American Physiological Society Undergraduate Research Excellence Fellowship (APS UGREF); Foundation (501c3); Metabolic Therapy and Cancer Research Account (#250244); Scivation: Florida High Tech Corridor Funding (#MED052‐0061361).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B56: Potential biomarkers for personalized oncology radiation in uterine cervical cancer

Abstract Uterine cervical cancer (UCC) is one of the most prevalent malignant neoplasms in the world. UCC develops beyond the stage in situ and is frequently treated by a combination of intracavitary radiation therapy and external beam radiation therapy; 30-40% of patients with similar prognosis factors do not respond equally to a comparable standard treatment. Therefore, the study and identification of prognostic biomarkers, which indicate the probable course of the disease in an untreated individual, and predictive biomarkers, which allow identification of subpopulations of patients most likely to respond to a given therapy, would be extremely useful in the selection of patients for the development of innovative and effective therapies for locally advanced, metastatic, and refractory uterine cervical cancer. Within work that we have been developing, we reported that gene expression of IGF1R is a strong predictive marker for lack of response to radiotherapy (p=0.018, 95% CI (1.7-41.2)), and patients HPV16 (+) have 28.6 times higher risk of failure treatment; consequently, with the present study, we proposed to determine whether expression of IGF-IR, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, HKII, and clinic-pathologic parameters can be used as prognostic and predictive biomarkers and as possible molecular targets for individualized treatment. Patients and Methods: This prospective cohort study included 149 patients with squamous cell carcinomas of the uterine cervix in FIGO stages IIB (n= 53) and IIIB (n=96). Of the 149 patients, 61 were treated with radiotherapy and 88 with concurrent radio-chemotherapy. Expression of the proteins CAIX, GLUT-1, HIF1α, HKII, IGF-IRα, IGF-IRβ, and Survivin was determined by immunohistochemistry in biopsies taken before treatment. Results: An overexpression was found for GAPDH (100%), followed by Survivin (87%), IGF-IRα (76.5%), IGF-IRβ (74.5%), HIF1α (74.1%), and IGF-IRα and IGF-IRβ (73%); strong expression was observed with low frequency for GLUT-1 (31.1%), CAIX (16.2%), and HKII (10.6%). Hgb level was significantly correlated with treatment response (p=0.01). With a median follow-up of 2.1 years, OS was decreased for patients overexpressing IGF-1R β (p=0.04). The OS of the subgroup of patients with anemia (Hgb &amp;lt; 11 g/dL) and concomitantly overexpressing IGF-1R and GLUT-1 was significantly decreased compared to the opposite control group (p=0.015). Conclusions: The expression of GLUT-1, IGF-1R, and Hgb (≤ 11 g/dl) is associated with poor prognosis, and thus appear to be interesting biomarkers of radiation resistance. In order to contribute to appropriate therapeutic management as a personalized neoadjuvant treatment, a prospective phase II study should be designed in the near future to evaluate the effect of turmeric (curcumin) as an inhibitor of IGF-1Rβ and GLUT1. Note: This abstract was not presented at the conference. Citation Format: Pablo Moreno-Acosta, Schyrly Carrillo, Oscar Gamboa, Alfredo Romero-Rojas, Jinneth Acosta, Diana Mayorga, Mónica Molano, Martha Cotes, Nicolas Magne. Potential biomarkers for personalized oncology radiation in uterine cervical cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B56.

Abstract B25: Meformin and LY294002 treatment regulate angiogenesis process through PI3K-MAPK/AKT/mTOR signaling pathways in mammary tumors

Abstract Introduction: The angiogenesis process is regulated by numerous factors, especially the hypoxia-inducible factor 1alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). The HIF-1 is a central regulator of pathophysiologic response of cells to hypoxia conditions, able to activate transcription of the gene that promotes the induction of VEGF, which in turn promotes angiogenesis through its ability to stimulate growth, migration, and invasion of endothelial cells, leading to the formation of new blood vessels and subsequent tumor growth. Metformin has demonstrated its ability to reduce the incidence of cancer in diabetic patients with positive results, especially in breast cancer, inhibiting cell growth by AMPK/Akt/mTOR signaling pathway. Likewise, LY294002, a PI3K signaling pathway inhibitor, has antiangiogenic features. In this context, the aim of this study was to evaluate the effectiveness of metformin and LY294002 inhibitor treatment in angiogenesis as a therapeutic strategy in mammary tumors in an in vitro and in vivo study. Materials and Methods: In the in vitro study, canine mammary tumor cell line (CF41) was cultured in DMEM high-glucose culture medium, at 37°C in 5% CO2. Cell viability was measured by MTT assay after treatment with different concentrations of metformin and LY294002 inhibitor. Once established the concentration of 5 mM for metformin and 5 µM of LY294002, the protein and gene expression of HIF-1alpha and VEGF were detected by immunocytochemistry and real-time PCR, respectively. For an in vivo study, CF41 were injected in nude athymic female mice and treated with metformin (200 mg/kg i.p.) for 4 weeks and LY294002 (7.5 mg/kg intratumorally) every 3 days for three times. At the end, mice were euthanized and the tumors were collected to determine the protein and gene expression of HIF-1alpha and VEGF and microvessel density by immunohistochemistry for CD31. The immunostaining was quantified by optical densitometry technique, slides were analyzed and photographed in Nikon Eclipse E200 microscope at 40X objective, and proteins were quantified by the ImageJ software analysis. For real-time PCR, the relative expression of the genes of interest was determined by DataAssist v3.0 software, using Ct method. Results: There was a significant decrease of cell viability after treatment with different concentrations of metformin and LY294002 in 24 hours. Both HIF-1alpha and VEGFA protein and gene expression significantly decreased after treatment with metformin and LY294002 or in combination. Furthermore, in animals, there was a decrease in tumor size, protein and gene expression of HIF-1alpha and VEGFA, in addition to a decrease of CD31 expression after all treatments. Conclusion: Our results suggest the potential effectiveness of metformin and LY294002 acting in angiogenesis process of mammary tumors. Financial support: FAPESP. Citation Format: Marina Gobbe Moschetta, Camila Leonel, Larissa Bazela Maschio-Signorini, Thaiz Ferraz Borin, Gabriela Bottaro Gelaleti, Lívia Carvalho Ferreira, Bruna Victorasso Jardim-Perassi, Nathália Martins Sonehara, Debora Aparecida Pires de Campos Zuccari. Meformin and LY294002 treatment regulate angiogenesis process through PI3K-MAPK/AKT/mTOR signaling pathways in mammary tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B25.