HIF-1alpha Expression Research Articles

121P - Influence of HIF-2alpha deregulation and overexpression of VEGF ligands on the response to aflibercept: Identification of predictive biomarkers

Background: Angiogenesis inhibitors are widely used for treatment of metastastic colorectal cancer. However, no predictive biomarkers are currently available for patient selection. Besides the tumor-associated endothelial cells, VEGF-signaling inhibitors target CRC cells that express VEGF as well as functional VEGFR1 receptors thereby mediating both paracrine and autocrine VEGF-signaling. The aims of this work is i) to establish the direct influence of aflibercept (Zaltrap®) that inhibits all three VEGFR1 ligands (VEGF-A, VEGF-B and PlGF) on CRC cells, ii) to identify tumor phenotypes associated with resistance to aflibercept in vitro and, iii) to extend these findings to human xenograft models. Methods: A panel of 12 well-characterized CRC cell lines was used to establish the influence of VEGFR1 stimulation (VEGF-A, VEGF-B, PlGF) and inhibition (aflibercept) on VEGF-mediated tumor cell migration. Expression of VEGF ligands and receptors was determined by qRT-PCR and ELISA assays. The in vivo influence of aflibercept was determined in human xenograft models and complemented by IHC and Western blot analysis. Results: Aflibercept inhibited the migration of most CRC cells under both normoxia and hypoxia including the highly sensitive HCT-116 cells. In contrast, LS174T cells did not respond to either aflibercept or to purified VEGF ligands. These cells expressed high levels of VEGF ligands and HIF2alpha, even under normoxia. Accordingly, aflibercept showed pronounced in vivo activity toward HCT-116 xenografts with 75% tumor growth inhibition but only 40% tumor growth inhibition toward LS174T tumors, compared to the corresponding vehicle controls. A similar trend was observed for bevacizumab with 41% tumor growth inhibition for HCT-116 and 32% inhibition for LS174T xenografts. IHC analysis of LS174T xenografts confirmed the strong expression of HIF2alpha and VEGF ligands as well as a modest inhibitory effect on the tumor-associated endothelia cells. Conclusions: We here report that aflibercept has direct antimigratory effects on most CRC cells. Strong expression of HIF-2alpha and VEGF ligands was accompanied by aflibercept resistance in vitro as in vivo. Legal entity responsible for the study: INSERM U938 and Université Pierre et Marie Curie (UPMC), Sorbonne Universités, Paris, France Funding: Sanofi-Genzyme Disclosure: A.K. Larsen: This work was supported in part by Sanofi Genzyme. E. Dochy: Employed by Sanofi-Genzyme. A. de Gramont: This research is in part supported by Sanofi. All other authors have declared no conflicts of interest.

Retraction: Hypoxia-Inducible Factor (HIF) 1α Accumulation and HIF Target Gene Expression Are Impaired after Induction of Endotoxin Tolerance.

The oxygen-sensitive transcription factor hypoxia-inducible factor 1 (HIF-1) is known as the key regulator of hypoxia-induced gene expression. In addition to hypoxia, endotoxins such as bacterial LPS as well as proinflammatory cytokines have been shown to induce HIF-1, suggesting an integrative role for HIF-1 in conditions of hypoxia and inflammation. Cells can become tolerant to endotoxins by repetitive exposure to LPS. Herein, we studied the effect of endotoxin tolerance on HIF-1alpha accumulation and expression of HIF target genes in human monocytic cells and primary mouse peritoneal macrophages. Tolerant cells had reduced levels of HIF-1alpha under hypoxia, which was due to lowered levels of HIF-1alpha mRNA. HIF-1alpha expression is under control of NF-kappaB and increased DNA binding of the p52 subunit of NF-kappaB was found in tolerant cells. Knock down of p52 abolished the effects of endotoxin tolerance on HIF-1alpha expression, which suggest a negative regulatory role of p52 on HIF-1alpha transcription during endotoxin tolerance. Endotoxin tolerant cells showed diminished expression of the HIF target genes phosphoglycerate kinase 1 and adrenomedullin and reduced viability under hypoxic conditions, as well as a significantly reduced invasion. Peritoneal macrophages from endotoxin-tolerant mice made showed significantly reduced HIF-1alpha protein accumulation and subsequent HIF target gene expression. We conclude that endotoxin tolerance impairs HIF-1alpha induction which reduces the ability of monocytic cells to survive and function under hypoxic conditions.

Abstract 891: Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription

Abstract Purpose: We previously revealed that the expression of Annexin A2 (ANXA2) was increased in ESCC by two-dimensional electrophoresis and MALDI-TOF and LC-ESI-IT MS. This study aims to investigated the implication of Annexin A2 in ESCC. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription. Experimental Design: Immunohistochemistry was performed to analyze the expression of ANXA2 in esophageal squamous cell carcinomas (ESCC). siRNA knockdown, transwell assay and wound-healing assays were used to investigate the function of ANXA2 in cells. Quantitative reverse-transcription polymerase chain reaction, Western blot analysis were used to seek the potential effector molecule of ANXA2. Chromatin immunoprecipitation, and reporter gene assays were used to confirm the relationship between c-Myc and HIF-1 alpha. Co-immunoprecipitation assay was performed to demonstrate the relationship between ANXA2 and c-Myc. Immunofluorescence was used to reveal the location of ANXA2. Results: Knockdown of Annexin A2 significantly inhibited the migration and invasion of ESCC cells. At the molecular level, VEGF, MMP-2, MMP-9, p-MEK and p-ERK were down-regulated in ANXA2-siRNA cells. The HIF-1 alpha mRNA and protein levels were significantly lowered after knocked down the Annexin A2. HIF-1 alpha is the effector molecule of ANXA2 in ESCC cells, and that ANXA2 affects the expression of HIF-1 alpha at the transcription level because when knocking down HIF-1 alpha, the level of VEGF, MMP-2, MMP-9, p-MEK and p-ERK reduced as well. c-Myc is a transcription factor for HIF-1 alpha. ANXA2 has a direct protein interaction with c-Myc and knockdown of ANXA2 down-regulated the expression of c-Myc. The Tyr 24 phosphorylation form of Annexin A2 (Y24D-ANXA2) was located in the nucleus, and the non-phosphorylation form (Y24A-ANXA2), in the cytoplasm and cell membrane. Transfection of pcDNA3.1-ANXA2-Y24D enhanced the migration and invasion as compared with that of pcDNA3.1-ANXA2-Y24A in esophageal cancer cells. A treatment with the Annexin A2 phosphorylation inhibitor, Dasatinib, changed the location of Annexin A2 and lowered the expression of c-myc and HIF-1 alpha. Conclusions: These results suggest that in esophageal cancer cells, phosphorylated Annexin A2 could enter into nucleus, which stabilizes c-myc and promotes the transcription of HIF-1 alpha, and the activates MAPK pathway, increases matrix metalloproteinase (MMP) activity and vascular endothelial growth factor (VEGF) expression, which results in the enhancement of cell invasion and migration. Citation Format: Sai Ma, Yan-Yi Jiang, Li-Fei Wu, Jia-Jie Hao, Yu Zhang, Xin Xu, Yan Cai, Ming-Rong Wang. Annexin A2 (ANXA2) promotes migration and invasion of esophageal cancer cells via stabilizing c-Myc and promoting HIF-1α transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 891. doi:10.1158/1538-7445.AM2017-891

Abstract 2056: MYC and HIF-2alpha mediates resistance to Epidermal Growth Factor Receptor (EGFR) antagonism in oral squamous carcinoma cells

Abstract Background: Oral squamous cell carcinoma (OSCC) is a devastating disease. Recent findings suggest that the EGFR antagonist including erlotinib may induce tumor regression alone or in combination with chemo/radiation. Our preliminary clinical study conducted at KaviKrishna laboratory indicates that many poor patients are taking the highly expensive drug Erlotinib, and or equivalent EGFR antagonists. However, oral cancer lesions are often hypoxic, and the hypoxia-induced cellular mechanisms might contribute to drug resistance. Here, we investigated, whether resistance to EGFR antagonists may involve MYC and HIF-2alpha, two transcription factors upregulated during hypoxia by using established oral cancer cell lines. We have also obtained primary oral cancer cells from patients living in the Kamrup district of Assam, where KaviKrishna laboratory is located. We intend to evaluate the drug sensitivity of these oral cancer cells towards EGFR antagonists. Method: We used SSC-25 and SCC-9 cell lines, as well patient derived primary oral cancer cells (n=5) for the study. Immunomagnetic sorting was performed to obtain ABCG2+ population. The self-renewal was studied using in vitro clonogenic and in vivo serial transplantation assay in NOD/SCID mice. Results: First, we identified a rare ABCG2+ expressing, highly tumorigenic cell population in SSC-25, and SCC-9 having cancer stem cell (CSC) like characteristics. Second, we found that the ABCG2+ cells exhibited sensitivity to PD158780 (10 uM; 62% inhibition within 48 hours), and AG1478 (10 uM; 56% inhibition within 48 hours), two small molecular inhibitors of EGFR tyrosine kinase. These small molecular inhibitors significantly inhibited the clonogenic capacity of the ABCG2+ cells. Next, we found that ABCG2+ cells also showed sensitivity to Erlotinib in the in vitro clonogenic assay. Third, the ABCG2+ cells, when exposed to hypoxia (<0.1% O2, 24 hours), exhibited enhanced expression and transcriptional activity of MYC, and HIF-2alpha. The post-hypoxia ABCG2+ cells exhibited complete resistance to PD158780, AG1478 and Erlotinib treatment, which could be reversed by siRNA silencing of MYC and or HIF-2alpha. ChIP assay revealed that HIF-2alpha directly binds to MYC in ABCG2+ cells. We found similar results in ABCG2+ cells obtained from primary oral cancer samples (n=4). Importantly, ABCG2+ cells directly isolated from patients exhibited hypoxic phenotype, including the high expression of HIF-1alpha, and HIF-2alpha, as well as resistance to erlotinib. Furthermore, erlotinib enhanced the stemness of ABCG2+ cells by activation of the MYC/HIF-2alpha self-renewal pathway (1). Conclusion: These data indicate that MYC and HIF-2alpha co-operate to mediate intrinsic resistance of oral squamous cancer cells to EGFR antagonist in the hypoxia microenvironment. (1). Bhuyan et al. Cancer Research, volume 76 (14), abstract 935; 2016 Note: This abstract was not presented at the meeting. Citation Format: Sukanya Gayan, Reza Bayat-Mokhtari, Bidisha Pal, Anupam Sarma, Joyeeta Talkudar, Sorra Sandhya, Rashmi Bhuyan, Seema Bhuyan, Jaishree Garhyan, Debabrat Baishya, Amal Kataki, Rakesh Bhatnagar, Herman Yeger, Bikul Das. MYC and HIF-2alpha mediates resistance to Epidermal Growth Factor Receptor (EGFR) antagonism in oral squamous carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2056. doi:10.1158/1538-7445.AM2017-2056

Acute hypoxic stress: Effect on blood parameters, antioxidant enzymes, and expression of HIF-1alpha and GLUT-1 genes in largemouth bass (Micropterus salmoides)

Dissolved oxygen (DO) plays a crucial role in survival, growth, and normal physiological functions of aquatic organisms. Nevertheless, the mechanisms involved in hypoxic stress and adaptation have not been fully elucidated in Largemouth bass (Micropterus salmoides). To reveal the effect of acute hypoxia on Largemouth bass, we simulated acute hypoxia (DO: 1.2 ± 0.2 mg/L) in the laboratory and analyzed physiological parameters (RBCs, Hb, SOD, CAT, NA+/K+-ATPase, GPx, and MDA) and gene expression (HIF-1alpha and GLUT-1) in Largemouth bass exposed to various durations of acute hypoxia (0, 1, 2, 4, 8, 12, and 24 h). Our results indicated that acute hypoxic exposure significantly increased RBCs but decreased Hb. In addition, antioxidant enzyme activity was enhanced significantly in the liver and muscles at the initial stage of acute hypoxic exposure, but decreased significantly in gills during the entire process of hypoxic exposure. Furthermore, the expression levels of HIF-1alpha and GLUT-1 mRNA were significantly up-regulated in Largemouth bass under acute hypoxic exposure. In conclusion, our study provides a valuable basis for further elucidation of hypoxic adaptation and facilitates husbandry for an economically valuable species.

Study on resurrection callus culture extract on the expression and mechanism of AQP-1 in Schwann cells after injury of the resurrection

Objective:To investigate the mechanism of AQP1 in Schwann cells and repair effect of resurrection callus culture extract on hypoxic injury cells. Method:Being cultured for 24 h, the morphological changes of the cells with different concentrations of CoCl2 were observed under the microscope and the changes of AQP1 were detected by Western blotting. Then being cultured for 24 h, the cells with different concentrations of esurrection callus culture extract were detected by CCK-8. By selecting the best concentration of resurrection callus culture extract and CoCl2, the experiment has to be continued. The changes of AQP1 and HIF-1 alpha protein were detected by Western blotting in 7 days. Result:6 μl/ml CoCl2 was selected as the optimal concentration of hypoxia model, not only because of the obvious changes in cell morphology, but no significant inhibition of cell growth, and the highest expression of AQP1. The optimum concentration of 2.5 μl/ml resurrection callus culture extract could significantly promote cell proliferation. Schwann cells increased expression of AQP1 induced expression of HIF-1 alpha, and resurrection callus culture extract can reduce AQP1 and HIF-1 alpha. Conclusion:There was a significant correlation between the expression of AQP1 and peripheral nerve Schwann cell edema, the expression of AQP1 was induced by HIF-1 after cells hypoxia. The resurrection callus culture extract can significantly decrease the expression of AQP1 and to alleviate edema in Schwann cells.

Abstract P2-05-21: Predictive value of quantitative HER2 and HER3 levels combined with downstream signaling markers in HER2-positive advanced breast cancer patients treated with lapatinib

Abstract Background: Clinical correlates of lapatinib resistance have not been well defined. Previous studies implicated genes regulated by the estrogen receptor (ER) and activation or mutation of proteins downstream from HER family receptors. In the current study, HER2 and HER3 expression levels were quantitatively measured using a VeraTag® fluorescence-based assay, in addition to seven downstream signaling proteins determined by IHC. All biomarkers were correlated with overall survival (OS) in patients treated with lapatinib. Methods: Formalin-fixed, paraffin-embedded samples were obtained from the primary tumor of 191 patients treated with lapatinib plus capecitabine following progression on trastuzumab. The HERmark® Breast Cancer Assay (Monogram Biosciences, South San Francisco) was used to quantify HER2 protein expression levels. HER3 protein expression was quantified using the VeraTag® technology (Monogram Biosciences). Expression of ER, PTEN, Cyclin E, HIF-2alpha, p-p70S6K, p-AMPK and p-MAPK were determined by IHC (Duchnowska et al., Oncotarget 2016; 7:550). OS analyses of HER2 and HER3 were stratified by key clinical variables, including stage and presence of a brain metastasis prior to lapatinib-based therapy. Results: Among the downstream signaling molecules, HIF-2alpha (r = -0.23; p = 0.047) and ER (r = -0.27; p = 0.005) were negatively correlated with HER2 expression after adjustment for multiple testing. PTEN appeared to correlate with HER3, but was not significant after adjustment for multiple testing. OS was significantly shorter for both those below the cut-off level of positivity by the HERmark assay (HR = 1.8; p = 0.029), and those with above median HER2 levels (HR = 1.7; p = 0.009), as compared to cases with in between levels. The relationship between HER2 and OS is also captured by a U-shaped, parabolic function in HER2 (p = 0.005). Elevated HER3 showed a trend toward a correlation with longer OS (HR = 0.66/log; p = 0.16), somewhat stronger in the ER-negative subset (HR = 0.55/log; p = 0.085) and in the subset with above-median HER2 (0.48/log; p = 0.10), where inhibiting HER2 activation of HER3 may be more important. In multivariate Cox models, HER2 (parabola, intermediate HER2 best, p = 0.001), presence of brain metastases (HR = 2; p < 0.001), ER (HR = 0.60; p = 0.009) and either p-p70S6K (HR = 0.66; p = 0.018) or p-AMPK (HR = 0.67; p = 0.022) were significantly associated with OS (p-p70S6K and p-AMPK were mutually correlated). Conclusions: Patients with moderately increased HER2 levels may have best outcomes while receiving lapatinib following progression on trastuzumab. This supports recent findings of a less benefit from lapatinib in patients with high HER2 expression (Nunciforo et al., SABCS 2015, P3-07-08). HER3 levels do not seem to substantially impact the prognosis. Further studies are warranted to explore the predictive utility of quantitative HER2 and HER3 in guiding HER2-directed therapies. Citation Format: Duchnowska R, Sperinde J, Czartoryska-Arlukowicz B, Mysliwiec P, Winslow J, Radecka B, Petropoulos C, Demlova R, Orlikowska M, Kowalczyk A, Lang I, Ziólkowska B, Debska-Szmich S, Merdalska M, Grela-Wojewoda A, Zawrocki A, Biernat W, Huang W, Jassem J. Predictive value of quantitative HER2 and HER3 levels combined with downstream signaling markers in HER2-positive advanced breast cancer patients treated with lapatinib [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-21.

Quantification of VEGF and Screening of Transcription Factor HIF-1α in Synovial Fluid of Polyarthritic Patients: Targets for Potential Anti-angiogenic Molecules

Rheumatoid arthritis (RA) is a polyarticular inflammatory autoimmune disease and osteoarthritis (OA) is an inflammatory degenerative disease of the articular cartilage. The current study efforts have been made to understand the relationship between the angiogenic genes VEGF, its regulator HIF-1α and MVD in RA and OA. The level of VEGF in serum and synovial fluid was increased in both RA and OA, but the levels were slightly more in cases of RA compared to that of OA. A corresponding increase in expression of HIF-1α and MVD counts was also noted in both conditions. Similar to VEGF levels, the expression of HIF-1α , MVD and CD-31 counts was higher among RA patients. These results thus indicate that there is a significant association between the angiogenic peptide VEGF in serum and synovial fluids, expression levels of transcription factor HIF-1α and microvessel density in the synovium of polyarthritic patients. The increased expression and regulation of VEGF, HIF-1alpha and MVD in these chronic inflammatory conditions are not only early indicators but also potential targets for antiangiogenic factors in the therapeutics of novel drugs for these smoldering conditions.

Abstract B22: Invitro and invivo proof of concept for an effective antineoplastic combination of novel anti HIF therapy in pancreatic cancer

Abstract New therapeutic approaches for pancreatic cancer are needed owing to the extremely poor prognosis, in large part as a consequence of high rates of metastasis. Hypoxia plays a crucial role in carcinogenesis, and metastasis. We also know that HIF plays a significant role in tumor resistance to cytotoxic therapies, and further explains the pancreatic cancer lack of response in clinic. Unfortunately to our knowledge there has been no effective therapy to this date to overcome such cellular pathology. To address this shortcoming, we used both in vitro and in vivo approaches to evaluate the overall effects of anti HIF-1alpha protocol in pancreatic cancer. Here we used a novel combination of two epigenetic modifiers that were investigated in 2 and 3 dimentional cell cultures to synergistically inhibit HIF, migration and invasion of pancreatic cancer cell lines, and further were able to show significant clinical application of such therapy in a series of metastatic or refractory disease in human. Background: Hypoxia-inducible factor-1alpha (HIF-1alpha) is essential for the pancreatic cancer progression and metastasis, and has been suggested to be a target for cancer therapy, as the result, there has been tremendous efforts in recent years to target HIF, as a dominant factor driving the metastatic progression of pancreatic cancer, a meaningful therapeutic approach, both independently as well as in combination with current standard of care. Both in vitro and in vivo analysis has suggested that hypoxia significantly promotes cell proliferation and migration, resulting in metastasis. Pancreatic cancer cells in which HIF-1alpha expression was inhibited were less invasive, with reduced resistance to hypoxia, impaired migration, and reduced capacity to cause metastasis. It is also suggested by our colleagues as well as our own published data, that epigenetic modifiers are able to improve the degradation of HIF through VHL. We were convinced that such approach we would be able to reduce the activity of HIF or hypoxia response elements ( HRE). We tested this hypothesis by application of a combination of epigenetic modifiers in laboratory and further in human, successfully. Methods: To illustrate the role of hypoxia-inducible factor-1alpha in pancreatic cancer metastasis and the value of the molecule as a target for pancreatic cancer therapy, we tested the pancreatic cell lines ( Panc 1) in the experimental therapeutic laboratory using a 3 dimentional cell culture model, and were able to show the significant spheroid production by these cells as a result on intratumoral hypoxia and HIF. Further we were able to test the HIF activity/Hypoxia response element ( HRE), after exposure to two synergistic compounds. It was shown that HIF activity significantly reduces by such combination, as a result of sensitization of cells to the subsequent exposure. Clinical application of such method showed significant clinical superiority to the standard of care alone. We will review a case series of patients with advanced disease, and their clinical outcome in summary. Conclusions: HIF-1alpha is a dominant factor driving the metastatic progression of pancreatic cancer is a potent therapeutic target for the disease. Here we were able to show synergism in combining epigenetic modifiers to inhibit HIF both invitro and in human, with relevant and significant outcome. We conclude that a multitargeted epigenetic therapy ( MTET) using combination of drugs modifying the HIF activity is clinically relevant and we further suggest large clinical trials to optimize the response in clinic. Citation Format: M. A. Nezami, Steve Hager.{Authors}. Invitro and invivo proof of concept for an effective antineoplastic combination of novel anti HIF therapy in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B22.

Implication of VHL, ERK5, and HIF-1alpha in clear cell renal cell carcinoma: Molecular basis

ObjectivesTo determine the expression status of several proteins related to VHL gene function and its relationship with common clinicopathological parameters. Material and methodsObservational, analytical, cross-sectional study with 50 patients diagnosed with clear cell renal cell carcinoma. The study analyzed VHL mutations and hypermethylation as well as protein expression of VHL, CA-IX, HIF-1alpha, VEGF, ERK1/2, and ERK5, relating them to clinical variables.A bivariate and multivariate descriptive logistical regression analysis was performed, using the presence of metastasis at diagnosis as dependent variable. ResultsThe study identified 13 (26%) VHL mutations related to nuclear grade (P = 0.036). VHL hypermethylation was found in 20% of cases. VHL expression was associated with the presence of mutations (P = 0.013), and the absence of expression was associated with nuclear grade and the presence of metastasis (P<0.05). HIF-1alpha was negative in only 5 cases. Vascular endothelial growth factor (VEGF) was positive in 31 of 47 cases and was associated with Fuhrman nuclear grade, presence of metastasis, and stage (P<0.05). ERK5 expression was increased in 58% of cases and associated with the presence of metastasis and more advanced stages (P<0.05). In the logistic regression analysis, the only variable remaining in the model was VEGF expression (P = 0.014). ConclusionsVEGF has prognostic value in clear cell renal cell carcinoma, and ERK5 may be a new prognostic marker in this type of tumor owing to its relationship with metastasis and more advanced stages.

Abstract 435: Prognostic biomarkers as molecular targets for individualized neoadjuvant treatment for cervical cancer

Abstract Cervical cancer is one of the most prevalent malignancy and of higher mortality in the world, and is considered a marker of underdevelopment. Conventional radiotherapy is one of the treatments used for this type of cancer. 30 to 40% of patients with similar prognosis factors not respond equally to a comparable standard treatment. The poor response to radiotherapy leads to the development of innovative and effective therapies for cervical cancer locally advanced, metastatic and refractory. A comparative analysis of cervical cancer in the context of other cancers may reveal that it is relatively smaller number of targeted molecular agents that have been tested. Accordingly, a number of biological agents are currently in clinical development for the purpose of, inhibiting angiogenesis, molecularly address EGFR and IGF-1R, modulation of cell cycle, of histone deacetylases, COX-2, mTOR and tumor microenvironment (hypoxia and glycolysis). Within work that we have been developing, reported that gene expression of IGF1R is a strong predictive marker for lack of response to radiotherapy, patients have 28.6 times higher risk of failure treatment; Objective: To determine whether expression of IGF-IR, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, HKII and clinicopathological parameters can be used as prognostic biomarkers to treatment outcome and as possible molecular targets. Patients &amp; Methods: This prospective cohort study included 149 patients with squamous cell carcinomas of the uterine cervix in FIGO stages IIB and IIIB between 2008 and 2011. The mean age was 46 years. Of the 149 patients, 61 were treated with radiotherapy and 88 with concurrent radiochemotherapy. 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: The highest increase was found in expression of GAPDH (100%), Survivin (87%), followed of, IGF-IRα (76.5%), IGF-IRβ (74.5%), IGF-IRα and IGF-IRβ concordance in the expression(73%), HIF1α (74.1%); strong expression was observed with low frequency for GLUT-1 (31.1%), CAIX (16.2%), HKII (10.6%). We found that patients who do not express IGF-1Rß, GLUT1 and having hemoglobin levels &amp;gt; 11g/dl have improved overall survival compared to those that express IGF-1Rß, GLUT1 and having hemoglobin ≤ 11g/dl (P = 0.0158). Conclusions: Using the expression of GLUT1, IGF-1Rß and Hb levels (≤ 11g/dl) as therapeutic molecular targets could contribute to an appropriate therapeutic management as individualized neoadjuvant treatment for cervical cancer Citation Format: Pablo Moreno-Acosta, Oscar Gamboa, Diana Mayorga, Alfredo Romero, Jinneth Acosta, Maria Carolina Sanabria, Martha Cotes Mestre, Nicolas Magne. Prognostic biomarkers as molecular targets for individualized neoadjuvant treatment for cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 435.

Abstract 920: Migratory cancer side population cells induces stem cell altruism in bone marrow mesenchymal stem cells to resist therapy, and enhance tumorigenic potential of non-tumorigenic cells

Abstract Stem cell may exhibit altruistic behavior that may benefit cancer cells. We recently demonstrated altruistic phenotype in human embryonic stem cells (Das B et al. Stem Cells 2012). The phenotype exhibited reversible induction of high HIF-2alpha and low p53 expression, associated with high glutathione secretion. We speculated that cancer stem cell might induce a similar altruistic phenotype in human bone marrow (BM) derived stem cells (hematopoietic, mesenchymal and endothelial cells). The altruistic reprogrammed BM cells may then facilitate tumor growth, as well as resist the toxicity of oxidative-stress inducing anti-cancer agents. To investigate this possibility, we have obtained conditioned media (CM) from migratory side-population (SPm) and non-SP cells of a diverse panel of tumors including epithelial tumors. The SPm cells exhibit very high tumorigenic capacity (Das B et al, Stem Cell, 2008). The CM was added to in vitro bone marrow (BM) derived CD133+ cells that contain hematopoietic, endothelial, and mesenchymal stem cells. We found that SPm derived CM (henceforth known as SPm-CM) treatment increased the self-renewal capacity of CD271+/CD45- BM-MSCs. Importantly, the reprogrammed CD271+ BM-MSCs (henceforth known as R-MSCs) phenotype exhibited enhanced stemness reprogramming, a cytoprotective mechanism associated with stem cell altruism (Das B et al. Stem Cells, 2012). In contrast, the treatment with CM obtained from non-SP cells did not exhibit R-MSCs. We found that VEGF/VEGR1 autocrine signaling may be involved in R-MSCs mechanism. Importantly, the R-MSCs derived CM reprogrammed non-CSCs to CSCs, and reduced the toxicity of chemotherapy on non-SP cells. In vivo, R-MSCs derived CM, when injected to mice, exhibited mobilization of CD271+ BM-MSCs to circulation. The circulatory CD271+ BM-MSCs exhibited distinct phenotype of R-MSCs including high expression of HIF-2alpha, and VEGFR1. Finally, in human cancer patients, we identified R-MSC phenotype in the peripheral circulation. These studies suggest that cancer stem cells may exploit stem cell altruism to reprogram BM-MSCs for their own benefit. The reprogrammed BM-MSCs gene expression may have the potential as a diagnostic marker for CSC-induced stem cell altruism. Citation Format: Joyeeta Talukdar, Rashmi Bhuyan, Jaishree Garhyan, Bidisha Pal, Sora Sandhya, Sukanya Gayan, Anupam Sarma, Reza Bayat-Mokhtari, Hong Li, Jyotirmoy Phukan, Wael Tasabehji, Seema Bhuyan, Amal Ch Kataki, Rika Tsuchida, Herman Yeger, Debabrata Baishya, Bikul Das. Migratory cancer side population cells induces stem cell altruism in bone marrow mesenchymal stem cells to resist therapy, and enhance tumorigenic potential of non-tumorigenic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 920.

Expression of HIF-1 alpha and its target genes in the Nanorana parkeri heart: Implications for high altitude adaptation

Expression of HIF-1 alpha and its target genes in the Nanorana parkeri heart: Implications for high altitude adaptation

Abstract A47: A microRNA signature identifies subtypes of triple-negative breast cancer and reveals miR-342-3p as regulator of a lactate metabolic pathway through silencing monocarboxylate transporter 1

Abstract Triple negative breast cancer (TNBC) represents a challenging tumor type due to their poor prognosis and limited treatment options. It is well recognize that clinical and molecular heterogeneity of TNBC is driven in part by post-transcriptional regulators such as miRNAs. To stratify TNBCs, we profiled 1050 miRNAs in 132 adjuvant TNBC tumors and 40 tumors from other immunophenotypes using an Affymetrix microarray platform. A NMF clustering analysis allowed us to identify 4 TNBC subtypes featuring unique miRNA expression patterns, disease free and overall survival rates and particular gene ontology enrichments (performed with GSEA algorithm). Our agglomerative approach was cross-validated by using two other clustering algorithms (k-means and consensus clustering). TNBC miRNAs subgroups were also correlated with the Lehmann intrinsic subtypes, finding a significant enrichment of immmnomodulartory and basal 1 subtypes in our high risk miRNA subgroups. 3 cell line models (MDA MB 468, MDA MB 231 and HS578T) were classified according to our miRNA signature, recapitulating two different miRNA subgroups. The TNBC tumors were compared against other phenotypes identifying differentially expressed miRNAs that together with the altered miRNAs within the subgroups allowed us to define interesting miRNAs for further functional analysis. We found low expression levels of miR-342-3p in TNBC tumors compared with other breast cancer phenotypes, and this down-regulation characterizes one of our miRNA subgroups with high risk to relapse. To characterize the functional its functional role, miR-342-3p was transiently transfected in the cell line MDA-MB-468, showing a decrease in cell proliferation, viability and migration rates. A gene expression profile (Human gene st, Affymetrix) revealed 140 altered mRNAs, from which 35 are potential direct targets of miR-342-3p defined by an in-silico analysis. The monocarboxylate transporter 1(MCT1), was confirmed as one target of miR-342-3p by a luciferase assay and western blot analysis. MCT1 repression by the miRNA promotes lactate efflux changes in the tumor cells, reflected in the accumulation of exogenous lactate and the increase in levels of extracellular endogenous lactate together with a decrease level of intra and inter cellular glucose concentration. We also explored other mechanisms that can contribute to the modulation of lactate and glucose levels such as the expression of MCT4, HIF1alpha and Glut1, but not significant changes were observed, pointing out the important role of the regulatory circuit of miR-342-3p and MCT1 in a particular TNBC tumor cells metabolism. These data suggest a metabolic change that favors a more glycolytic environment, which lead to a glucose deprivation context that may contribute to the reduction in proliferation, viability and migration capabilities already described. Furthermore, to define the main transcriptional modulator of the intronic miR-342-3p in TNBC, we evaluated the correlation between the expression of estrogen receptor, miR-342-3p and its host gene EVL in TNBC tumors from TCGA data. A significant correlation between these transcripts was observed. These results, together with information from other studies and Chip-seq analysis suggest that the ER activates the transcription of EVL and consequently the intronic miR-342-3p. We validated these correlations in our cellular model by transiently silencing of the ER. Our data provides evidence that miRNAs sub-classification signature can recapitulate the biological and clinical heterogeneity of TNBC. Moreover, show that miRNAs regulate important oncogenic pathways in TNBC cells such as: proliferation, cell movement and apoptosis in the different subgroups. Citation Format: Sandra L. Romero-Cordoba, Sergio Rodriguez-Cuevas, Rosa Rebollar-Vega, Veronica Bautista-Pina, Antonio Maffuz-Aziz, Elda Tagliabue, Marilena Iorio, Elvira D'Ippolito, Sara Baroni, Ilaria Plantamura, Alfredo Hidalgo-Miranda. A microRNA signature identifies subtypes of triple-negative breast cancer and reveals miR-342-3p as regulator of a lactate metabolic pathway through silencing monocarboxylate transporter 1. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A47.

PLUTO: A randomised phase II study of pazopanib versus paclitaxel in relapsed urothelial tumours.

430 Background: Two previous single arm trials have drawn conflicting conclusions regarding pazopanib in urothelial bladder cancers (UBC) after failure of platinum-based chemotherapy. PLUTO is a randomized phase II trial to compare the efficacy of pazopanib with paclitaxel in this setting. Methods: It was planned for 140 patients with progressive, advanced UBC (with at least a component of transitional cell carcinoma) who had previously received a single prior platinum containing chemotherapy regimen for advanced UBC to be randomised(1:1). Patients in arm A received weekly paclitaxel 80mg/m2 and those in arm B received pazopanib (800mg once daily) until progression. The primary endpoint was overall survival analysis of which would occur after 110 events. The efficacy was assessed by an independent data monitoring committee during the trial. Results: Between Aug 2012- Oct 2014, 131 patients were randomised. 74.0% of patients had visceral metastases. The study was terminated early by the IDMC due to futility of pazopanib. Final analysis after 110 deaths occurred in July 2015. The median number of cycles of paclitaxel received was 4 (12 weeks). The median time on pazopanib was 11 weeks. Median overall survival was 8.0 months for paclitaxel [80% confidence interval (CI) 6.9 to 9.7 months] and 4.7 months for pazopanib [80% CI 4.2 to 6.4 months]. The hazard ratio (HR) adjusted for baseline stratification factors was 1.25 [80% CI 0.96 to 1.63; 1-sided p = 0.86] in favour of paclitaxel. Median progression free survival was 3.2 months for paclitaxel [80% CI 2.9 to 5.0 months] and 3.1 months for pazopanib [80% CI 2.7 to 3.8 months]. The adjusted HR for PFS was 1.06 [80% CI 0.83 to 1.36; 1-sided p = 0.62] (in favour of paclitaxel). Discontinuations for toxicity occurred in 6.3% and 20.0% for paclitaxel and pazopanib respectively. VHL, HIF1alpha, VEGFR2 and PD-L1 expression were measured from archived tissue and correlated with outcome. Conclusions: Pazopanib does not have greater efficacy than paclitaxel in the second line treatment of UBC. There is a trend towards superior OS with paclitaxel. Clinical trial information: NCT00949455.

The Influence of HIF-1&amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt; Expression on Apoptosis and Number of T Lymphocyte in Peyer’s Patches after Burn with Delayed Fluid Resuscitation in Rats at Plateau

Objective: To research the expression of hypoxia inducible factor-1 alpha (HIF-1 alpha) on the apoptosis and number of T lymphocyte in Peyer’s patches after severe burn on plateau in rats. Methods: Wistar rats (n = 130) were subjected to deep thickness burn injury (30% TBSA, III degree), at two different altitudes. 60 of them were given delayed fluid resuscitation (DFR, n = 30 at each altitude) 6 h after burn at different altitude; 60 of them were carried out immediate fluid resuscitation (IFR, n = 30 at each altitude); 10 rats were subjected to 37°C warm water as sham burn (SG, n = 10). The Peyer’s patches were harvested from the ileum of rats at different time point after burn respectively. The expression of HIF-1 alpha, CD3(+) and the apoptosis and number of T lymphocyte in Peyer’s patches were detected by tissue microarray technology and immunohistochemistry. Results: The apoptosis was higher in DFR group than that in IFR group. The increase in HIF-1 alpha expression was observed mainly on cell nucleus in T lymphocytes. The expression levels of HIF-1 alpha in Peyer’s patches were much higher in DFR group and IFR group than those in SG, and they were higher at high altitude (3848 metres) than those at lower altitude (1517 metres), and also higher in DFR group compared with IFR group (all P + in Peyer’s patches were much lower in DFR group and IFR group than those in sham group, and the lowest value appeared at 12 hours after burn (all P

Effects of cobalt and chromium ions on HIF-1alpha expression and oxidative stress in macrophages in vitro

Effects of cobalt and chromium ions on HIF-1alpha expression and oxidative stress in macrophages in vitro

Role of epigenetic modulation in hypobaric hypoxia induced memory impairment

Event Abstract Back to Event Role of epigenetic modulation in hypobaric hypoxia induced memory impairment Rahul Kumar1, Vishal Jain2, Neetu Kushwah1, Aastha Dheer1, Dipti Prasad1 and Shashi B. Singh1* 1 Defense Institute of Physiology and Allied Sciences (DIPAS) DRDO, Neurobiology Department, India 2 Defense Institute of Physiology and Allied Sciences (DIPAS) DRDO, Neurophysiology Department, India Long term memory formation requires a series of cellular as well as molecular adaptation that control the gene expression at the transcription level. However, epigenetic mechanisms such as histone acetylation, methylation, phosphorylation and DNA methylation have emerge critical regulators of transcription during memory consolidation process. Epigenetic mechanism controls gene expression bidirectional i.e. either enhance or repress gene expression. In addition, histone acetylation has been the main mechanism of epigenetics and play important role in memory formation as well as reconsolidation. Stressful environmental conditions such as hypobaric hypoxia (HH) leads to change in learning and memory. Present study is designed to explore global as well as specific histone modifications, mainly acetylation in different brain regions during different duration of HH exposure. Sprague Dawley rats (220 to 230 gm) were exposed to different duration of HH i.e. 1, 3, 7 and 14 days in an animal decompression chamber at an altitude of 25,000 feet. After exposure, rats were sacrificed and hippocampus and cortex were isolated. Activity of histone acetylase (HAT), histone deacetylase (HDAC) and DNA methyl transferase (DNMT) was analyzed using kits. Further specific histone acetylation sites i.e. H2BK5Acetylation, H3K9Acetylation and H4K14Acetylation were also studied in different brain regions. Results from the present study revealed the correlation between expressions of HDAC and HIF-1 alpha which further control other hypoxia responsive genes in both the brain regions. Gradual increased expression of HDAC on HH exposure causes repression of learning and memory related genes. In addition, the decline in H2BK5Ac expression may also account for inactivation of transcription of memory responsive gene leading to decline in memory. In brief, it can be concluded that epigenetic modification at histone acetylation sites may account for HH mediated memory impairment. Keywords: Memory, epigenetic, in vivo, histone acetylation, hypobaric hypoxia Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kumar R, Jain V, Kushwah N, Dheer A, Prasad D and Singh SB (2016). Role of epigenetic modulation in hypobaric hypoxia induced memory impairment. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00191 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Shashi B Singh, Defense Institute of Physiology and Allied Sciences (DIPAS) DRDO, Neurobiology Department, New Delhi, North Delhi, India, drshashisingh@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rahul Kumar Vishal Jain Neetu Kushwah Aastha Dheer Dipti Prasad Shashi B Singh Google Rahul Kumar Vishal Jain Neetu Kushwah Aastha Dheer Dipti Prasad Shashi B Singh Google Scholar Rahul Kumar Vishal Jain Neetu Kushwah Aastha Dheer Dipti Prasad Shashi B Singh PubMed Rahul Kumar Vishal Jain Neetu Kushwah Aastha Dheer Dipti Prasad Shashi B Singh Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract A04: Is the lack of clinical success with antiangiogenic therapy due to vascular normalization not being a universal phenomenon?

Abstract Introduction: Pre-clinical studies have demonstrated that combining angiogenesis inhibitors (AIs) with conventional radiation and chemotherapy treatments can be an effective anti-tumor therapy. However, clinical benefits from such combinations have been more modest. A principal mechanism by which AIs are believed to increase tumor sensitivity to radiation and chemotherapy is vascular normalization, a process which should improve tumor blood perfusion and oxygenation. Here we review the universality of this concept by examining those pre-clinical studies that directly determined the effect of AI treatment on tumor oxygenation. Methods: This review was based on searching the PubMed database using the criteria angiogenesis inhibitors combined with one of each of the following: oxygenation, hypoxia, or normalization coupled with our own knowledge within the fields of tumor hypoxia and vascular targeting. A total of 51 pre-clinical studies were identified. The techniques used to monitor hypoxia/oxygenation included immunohistochemical analysis of hypoxia marker binding (pimonidazole and EF5) or hypoxia gene expression (CA9 and HIF-1alpha), direct oxygenation measurements (Eppendorf electrode, EPR oximetry, Oxylite probe or phosphorous quenching), or radiation response. The AIs included the two principal classes of inhibitors, those targeting VEGF (bevacizumab, DC101) and tyrosine kinase (vandetanib, sunitinib, sorafanib, axitinib, pazopanib, AG-013736, PTK787/ZK222584, SU5416), as well as a variety of other agents (TNP-470, suramin, endostatin, thrombospondin, thalidomide, anginex, arginine deiminase, nucleolin antagonist, nelfinavir). Results: 19 studies demonstrated significant improvements in tumor oxygenation status. These included a decrease in hypoxia marker labelling (bevacizumab, DC101, suramin, endostatin, nucleolin antagonist); an increase in oxygenation determined using the Eppendorf electrode (TNP-470 and an anti-VEGF antibody), EPR oximetry (thalidomide, vandetanib, SU5416), and Oxylite probe (bevacizumab); an increase in hypoxia regulated gene expression (endostatin and SU5416); and enhanced radiation response (thrombospondin). In contrast, 21 studies found a significant decrease in tumor oxygenation using hypoxic markers measured by immunohistochemistry (bevacizumab, DC101, sunitinib, pazopanib, AG-013736, nelfinavir) or PET activity (PTK787/ZK222584), and increased expression of HIF-1alpha (bevacizumab). Moreover, 9 studies reported no change in tumor oxygenation as measured with hypoxic markers (endostatin and SU5416), direct oxygen measurements (anti-VEGF antibody, DC101, arginine deiminase), or radiation response (DC101). Finally, two studies actually reported both increases and decreases when using direct oxygen measurements depending on the time after treatment with bevacizumab or DC101. Conclusions: While there is clear evidence that tumor oxygenation can improve following AI treatment, consistent with vascular normalization occurring, this is clearly not a universal finding. Importantly the effects on tumor hypoxia/oxygenation status show no relationship to either the specific AI or the assay used to monitor these parameters. These findings suggest that for the combination of AIs with conventional anti-cancer therapies to be most effective it will be important to establish the impact the AI has on the tumor vasculature and associated tumor pathophysiology in a given treatment setting. Citation Format: Michael R. Horsman, Dietmar W. Siemann. Is the lack of clinical success with antiangiogenic therapy due to vascular normalization not being a universal phenomenon? [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A04.

Bromoenol Lactone Attenuates Nicotine-Induced Breast Cancer Cell Proliferation and Migration.

ObjectivesCalcium independent group VIA phospholipase A2 (iPLA2β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA2β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined.Method and ResultsWe found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA2β dependent manner. These results suggest that iPLA2β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis.ConclusionThe present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA2β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis.