Manu Gnanamony

Chemoresistance in pancreatic cancer: Emerging concepts (Review)

Pancreatic cancer is one of the most lethal types of cancer in the world. The incidence of pancreatic cancer increases each year with no significant decrease in mortality. Pancreatic cancer is a complex disease, and this complexity is partly attributed to late diagnosis, an aggressive phenotype, environmental factors and lack of effective treatment options. Surgical resection followed by adjuvant chemotherapy is the treatment of choice for early stage cancer, whereas gemcitabine is the standard first line therapy for patients with advanced stage disease. Treatment regimens comprising folinic acid, 5-fluorouracil, irinotecan, oxaliplatin and nab-paclitaxel have demonstrated modest effects in improving median survival rates. A number of other chemotherapeutics are currently undergoing clinical trials as components of combination therapies with gemcitabine. An increasing number of novel molecular targets and cellular pathways are being identified, which highlights the complexity of this disease. The development of chemoresistance to gemcitabine is multifactorial and there exists an interplay between pancreatic cancer cells, the tumor microenvironment and cancer stem cells. These components appear to be governed by a complex network of non-coding RNAs such as micro RNAs and long non-coding RNAs. In the present study, studies describing previous research on the understanding of the factors associated with the development of chemoresistance to gemcitabine in pancreatic cancer are reviewed. A comprehensive understanding of the multiple pathways of chemoresistance is key to develop next generation therapeutics to pancreatic cancer.

The ‘SPARC’ of life: Analysis of the role of osteonectin/SPARC in pancreatic cancer (Review)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most clinically challenging cancers to manage. An estimated 48,960 people will be diagnosed with pancreatic cancer in 2015, of that population, 94% are projected to perish within 5 years. These dismal survival rates can be attributed, in part, to an advanced diagnosis occurring in 80% of cases. The heterogeneous and dynamic microenvironment of pancreatic cancer, and the lack of both specific risk factors and efficacious screening tools contribute to the challenge of diagnosing pancreatic cancer in its early stages. These clinical challenges have directed research into the unique characteristics that define PDAC. Recently, there has been an increased focus on the interaction of tumor cells with their microenvironment in the hope of identifying new therapeutic targets. One of the most promising avenues in this new vein of research is targeting protein communication between the cancer cells and the extracellular matrix. The secreted protein acidic and rich in cysteine (SPARC) is one such extracellular matrix protein that has shown potential as a therapeutic target due to its influence on PDAC invasion and metastasis. In this review, we discuss the complex interaction of SPARC with PDAC cells and its potential to guide treatment and eventually improve the survival of patients diagnosed with this devastating disease.

SPARC overexpression combined with radiation retards angiogenesis by suppressing VEGF-A via miR‑410 in human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and despite aggressive therapy survival rates remain low. One of the contributing factors for low survival rates is aggressive tumor angiogenesis, which is known to increase due to radiation, one of the standard therapies for neuroblastoma. Therefore, targeting tumor angiogenesis can be a viable add-on therapy for the treatment of neuroblastomas. In the present study, we demonstrate that overexpression of secreted protein acidic and rich in cysteine (SPARC) suppresses radiation induced angiogenesis in SK-N-BE(2) and NB1691 neuroblastoma cells. We observed that overexpression of SPARC in SK-N-BE(2) and NB1691 cells reduced radiation induced angiogenesis in an in vivo mouse dorsal skin model and an ex vivo chicken CAM (chorioallantoic-membrane) model and also reduced tumor size in subcutaneous mouse tumor models of NB. We also observed that SPARC overexpression reduces VEGF-A expression, in SK-N-BE(2) and NB1691 NB cells via miR-410, a VEGF-A targeting microRNA. SPARC overexpression alone or in combination with miR-410 and radiation was shown to be effective at reducing angiogenesis. Moreover, addition of miR-410 inhibitors reversed SPARC mediated inhibition of VEGF-A in NB1691 cells but not in SK-N-BE(2) NB cells. In conclusion, the present study demonstrates that the over-expression of SPARC in combination with radiation reduced tumor angiogenesis by downregulating VEGF-A via miR-410.

Targeting the Expression of Cathepsin B Using CRISPR/Cas9 System in Mammalian Cancer Cells

Cathepsin B belongs to a family of cathepsins and plays an important role in normal physiological functions in the cell. However, overexpression of cathepsin B has been associated with different malignancies, and this has made it an attractive pharmacological target. The advent of CRISPR-Cas9 technology has allowed researchers to efficiently knock down genes with very less nonspecific activity compared to earlier methods. The protocol described below will enable investigators to develop cathepsin B knockdown stable cells and explains ways to study the knockdown.

SPARC overexpression suppresses radiation-induced HSP27 and induces the collapse of mitochondrial Δψ in neuroblastoma cells

Neuroblastoma is the cause of >15% of cancer-associated mortality in children in the USA. Despite aggressive treatment regimens, the long-term survival rate for these children remains at <40%. The current study demonstrates that secreted protein acidic and rich in cysteine (SPARC) suppresses radiation-induced expression of heat shock protein 27 (HSP27) in vivo and suppresses mitochondrial membrane potential (Δψ) in neuroblastoma cells. In the present study, the overexpression of SPARC in SK-N-BE(2) and NB1691 neuroblastoma cell lines suppresses radiation-induced G2M cell cycle arrest, proliferation, HSP27 expression (in vitro and in vivo) and induces the collapse of the mitochondrial Δψ. Gene ontology analysis demonstrated that the overexpression of SPARC combined with irradiation, induces the expression of dissimilar molecular function genes in SK-N-BE(2) and NB1691 cells, providing evidence of a dissimilar response signaling pathway. These results demonstrate that overexpression of SPARC suppresses radiation-induced HSP27 expression in neuroblastoma cells and the combination of SPARC and radiation induces the expression of protein 21, but suppresses neuroblastoma tumor density in in vivo mouse models. SPARC also induces mitochondrial Δψ collapse in SK-N-BE(2) and NB1691 neuroblastoma cells.

Chronic radiation exposure of neuroblastoma cells reduces nMYC copy number

Neuroblastoma accounts for >15% of cancer-associated mortalities of children in the USA. Despite aggressive treatment regimens, the long-term survival for these children remains <40%. The identification of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (nMYC) gene amplification during diagnosis is associated with poor prognosis in neuroblastoma. There are limited studies examining changes in nMYC copy numbers in response to therapy and its biological effect on cancer cells. The aim of the present study was to evaluate the effect of radiation on nMYC expression and amplification status in high-risk neuroblastoma. The effect of acute (5 Gy) and chronic (25 Gy) radiation on two nMYC-amplified cell lines, SK-N-BE (2) and NB-1691, was investigated. The results demonstrate that, following chronic but not acute radiation, the two cell lines regained their proliferation potential similar to the controls. This increased proliferation was characterized by loss of nMYC mRNA and protein expression. It was also revealed that nMYC loss was accompanied by nuclear localization of c-Myc. Using fluorescent in situ hybridization and quantitative polymerase chain reaction analysis, the results of the present study demonstrated that chronic radiation causes a severe loss of nMYC gene copy number. The present study is the first to provide experimental evidence that prolonged radiation therapy affects nMYC gene copy number in high-risk neuroblastoma but does not significantly improve the prognostic outlook.

Abstract 2154: TX1111: a peptide homologue of Topoisomerase-1 sensitizes pancreatic cancer cells to gemcitabine

Pancreatic Ductal Adeno Carcinoma (PDAC) is the fourth most common cause of cancer deaths in the United States and accounts for over 95% of all pancreatic cancers. The combined 1- and 5-year survival rates for PDAC are very poor, at 25% and 6% respectively. A major hallmark of pancreatic cancer is tumor recurrence and extremely poor response to chemotherapy. The current standard of care for patients with advanced pancreatic cancer is a chemotherapy regimen that includes gemcitabine. This treatment results in a modest benefit i.e., an increase in survival of only 5 weeks. The high mortality in patients diagnosed with this pancreatic cancer is primarily due to its drug-resistant nature and the lack of effective therapeutic strategies to overcome drug resistance. This disappointing situation strongly suggests that an improved and increased understanding of drug resistance mechanisms could lead to the development of novel therapeutic strategies for the successful treatment of patients with pancreatic cancer. Our studies have shown that one of the contributing factors that lead to chemotherapy resistance is the protease activator urokinase plasminogen activator (uPA). To validate the contribution of uPA in chemoresistance, we overexpressed uPA in MIAPaCa-2 and PANC-1 cells and determined chemoresistance. We observed that uPA overexpressed cells showed increased chemoresistance in both Mia PaCa-2 (>25 fold) and PANC-1 (>6 fold). Mass-Spec analysis of nuclear uPA-IP from pancreatic cancer cells revealed that uPA interacts with Topoisomerase-1 (TOPO-1). This was further validated by western blot analysis of nuclear co-IP in pancreatic cancer cells. Further the binding domains of TOPO-1 to uPA were identified using overlapping a peptides array (PepSpot-JPT) followed by synthesis of these peptides. We observed that a TOPO-1 homologous peptide designated TX1111 was capable of significantly suppressing gemcitabine resistance in pancreatic cancer cells. Our observations show that the specific peptide TX1111 suppresses chemoresistance by 45% in Mia Paca-2 cells (p These results demonstrate that: 1. Overexpression of uPA promotes gemcitabine resistance in pancreatic cancer cells 2. uPA and TOPO-1 interact in the nucleus of pancreatic cancer cells 3. The peptide homologue of TOPO-1, TX1111 sensitizes gemcitabine resistant pancreatic cancer cells. Citation Format: Manu Gnanamony, Victoria Stepanova, Lily Criscione, Jerusha Boyineni, Stephen J. Marshall, AiXuan Holterman, Christopher S. Gondi. TX1111: a peptide homologue of Topoisomerase-1 sensitizes pancreatic cancer cells to gemcitabine. [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 2154.

Abstract 2480: Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma cells

Neuroblastoma is the most common extra-cranial solid tumor in children. Despite aggressive therapy prognosis of high risk neuroblastoma remains guarded and also unpredictable because of heterogeneity of tumor response to treatment. Radio-resistance has been recognized as a significant contributor to aggressive clinical behavior of neuroblastoma. Characterization of genetic alterations have been used for predicting clinical outcome in neuroblastomas and one of the most studied genetic markers in aggressive neuroblastoma is nMyc/MYCN. The MYCN gene belongs to the MYC family of transcription factors and nMyc expression is to a large extent inversely correlated with cMyc expression and nMyc expression is also associated poor clinical outcomes. In this study, we have attempted to correlate the expression of nMyc in neuroblastoma cells NB1691 and SKNBE2 to radiation resistance. We exposed neuroblastoma cells NB1691 and SKNBE2 to chronic radiation doses with a cumulative high energy X-ray radiation dose of 25Gy achieved by dosing at 5Gy increments over a period of 15 days. Cells surviving after the 25Gy cumulative radiation dose were selected and designated as NB1691R and SKNBE2R. Expression levels of nMyc, cMyc and uPA were determined. We observed that the radiation tolerant cells showed at least 164 fold increase in the expression of uPA in NB1691R cells and at least 97 fold increase in SKNBE2R cells. We also observed that the expression levels of nMyc decreased in both NB1691R and SKNBE2R cells to almost undetectable levels. Further, the expression levels of cMyc revealed that NB1691R cells showed a 208 fold increase and SKNBE2R a 71 fold increase when compared to parental cells. We further determined whether the expression of uPA can modulate the expression of nMyc. We silenced uPA expression in NB1691 cells and observed that downregulation of uPA moderately rescued nMyc expression. Cell cycle analysis revealed that acute radiation of NB1691 cells induced accumulation of cells in the G2/M phase and un-radiated NB1691R cells continued to show cells accumulated in the G2/M phase similar to acute radiated NB1691 cells. Our study shows that: 1. Chronic radiation exposure suppresses the expression of nMyc and induces the expression of cMyc. 2. cMyc expression was accompanied with the expression of uPA and suppression of uPA expression moderately rescued the expression of nMyc. Our preliminary findings suggest that chronic exposure to radiation alters the nMyc/cMyc ratio in neuroblastoma cells and induces the expression of the pro-invasive molecule uPA. Citation Format: Manu Gnanamony, David Pinson, Reuben Antony, Karen S. Fernandez, Julian Lin, Pushpa A. Joseph, Christopher S. Gondi. Chronic radiation exposure increases uPA and cMyc expression levels but decreases nMyc expression levels in neuroblastoma 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 2480.

Abstract 5213: RUNX2 modulates the angiogenic potential of human neuroblastoma cells

Neuroblastoma is the most common extracranial pediatric solid tumor with an undifferentiated status and characterized by heterogeneous clinical courses ranging from spontaneous regression to a very aggressive malignant progression. The RUNX gene family comprises RUNX1, RUNX2, and RUNX3 transcription factors and plays pivotal roles in development, differentiation and tumorigenesis. Hypoxia is known to be a significant physiological stress in the tumor microenvironment and is associated with tumor malignant phenotype. In this study, we examined the effects of hypoxia on the Runx2 expression in human neuroblastoma cells and the role of RUNX2 on angiogenic potential of neuroblastoma cells. Runx2 has two isoforms, Runx2-I and Runx2-II. Isoform I is controlled by the P2 proximal promoter whereas isoform II is transcribed from the P1 distal promoter. Increased expression of mRNAs of isoform I and II as well as intact isoform of RUNX2 was observed in NB1691 cells exposed to hypoxia. Furthermore, hypoxia enhanced the activity of a luciferase reporter containing Runx2 P1 promoter in NB1691 cells. Hypoxic exposure caused an increase in protein levels of RUNX2 as well as survivin, a RUNX2 downstream target gene in NB1691 cells. Hypoxia also stabilized RUNX2 protein levels in NB1691 cells without altering the stability of RUNX2 mRNA. Hypoxia enhanced angiogenic potential of NB1691 cells. Knockdown of Runx2 significantly decreased VEGF mRNA and protein in NB1691 cells exposed to hypoxia. A decrease in angiogenesis was observed in chorioallantoic membrane assay performed using conditioned medium collected from RUNX2 siRNA treated hypoxic NB1691 cells compared with control. Further, transcript levels were measured by PCR array of human angiogenesis pathway and the analysis showed down regulation of EDN1, FIGF, TEK and VEGF A mRNAs in hypoxic NB1691 cells treated with RUNX2 siRNA. In conclusion, our study suggests that hypoxia upregulates RUNX2 expression in NB1691 cells and knockdown of RUNX2 decreases the angiogenic potential of hypoxic NB1691 cells. Citation Format: Manu Gnanamony, Indra Mohanam, Sanjeeva Mohanam. RUNX2 modulates the angiogenic potential of human neuroblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5213. doi:10.1158/1538-7445.AM2015-5213

Abstract 499: RUNX2 protects human neuroblastoma cells against apoptosis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Neuroblastoma is the most common extracranial pediatric solid tumor with an undifferentiated status and is characterized by heterogeneous clinical courses. The RUNX gene family comprises RUNX1, RUNX2, and RUNX3 transcription factors and is closely involved in a variety of cellular processes including development, differentiation, and tumorigenesis. Since RUNX genes play crucial roles in neuronal differentiation, we studied the expression of RUNX mRNAs in human neuroblastoma NB1691 cells. An increased RUNX2 mRNA expression was found in NB1691 cells compared with RUNX1 and RUNX3 mRNAs. Hypoxia is known to be a significant physiological stress in the tumor microenvironment and is associated with tumor malignant phenotype. NB1691 cells exhibited an increased expression of RUNX2 mRNA upon exposure to hypoxia compared with normoxic cells. Knockdown of HIF-2α, but not HIF-1α, decreased hypoxia-induced up regulation of RUNX2 mRNA in NB1691 cells. BothHIF-1α and HIF-2α mRNAs as well as proteins were downregulated in hypoxic NB1691 cells treated with RUNX2 siRNA compared with non-targeted control siRNA. GLUT1, a HIF-1α downstream target gene and Oct4, a HIF-2α downstream target gene were also found to be decreased in RUNX2 siRNA-treated NB1691 cells exposed to hypoxia. Runx2 knockdown using RNA interference in NB1691 cells resulted in decreased cell proliferation under normoxic as well as hypoxic conditions. An increased percentage of apoptotic cells was found in NB1691 cells treated with RUNX2 siRNA, and this percentage further increased in hypoxic NB1691 cells treated with RUNX2 siRNA. Further, these observations were validated by TUNEL assay. Real-time PCR analysis revealed elevated levels of Bax, Puma, Noxa and Nix in NB1691 cells treated with RUNX2 siRNA compared with cells treated with non-targeted control siRNA-treated cells under hypoxic conditions. In conclusion, our study suggests that hypoxia up regulates RUNX2 in NB1691 cells in a HIF2α- dependent manner and RUNX2 protects neuroblastoma cells against apoptosis. Citation Format: Manu Gnanamony, Indra Mohanam, Sanjeeva Mohanam. RUNX2 protects human neuroblastoma cells against apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 499. doi:10.1158/1538-7445.AM2014-499