CONCLUSIONS STUDY DESIGN BACKGROUND Neuronal Pentraxin2: A Novel Tumor-Specific Molecular Target That Mediates Clear Cell Renal Cell Carcinoma Malignancy Christina A. von Roemeling 1 , Derek Radisky 1 , Laura Marlow 1 , Simon J. Cooper 1 , Stefan K Grebe 2 , Panagiotis Z. Anastasiadis 1 , Han W. Tun 1,3 , John A. Copland 1 1 Department of Cancer Biology, Mayo Clinic Florida, Jacksonville, Florida; 2 Laboratory Medicine and Pathology, Mayo Clinic Minnesota, Rochester, MN; 3 Department of Hematology/Oncology, Mayo Clinic Florida, Jacksonville, Florida April 7, 2014: Abstract LB-111 y CLINICAL IMPACT RESULTS REFERENCES A Figure 1: NPTX2 Expression Profile in ccRCC Figure 4: Loss of GluR4 Recapitulates NPTX2 Knockdown Phenotype D E C Translational Relevance: Patients with metastatic ccRCC have poor prognoses, with an estimated 5 year overall survival of less than 10%. This is due to lack of remedial therapies that produce durable disease stabilization or tumor regression. Drug resistance is a hallmark of ccRCC and is linked to cancer cell heterogeneity. Additionally, there is a lack of known molecular factors that can be targeted pharmacologically. The clear cell variant of RCC also frequently manifests as metastatic disease, likely due to its disposition for increased migratory capacity and the formation of undetectable micrometastases. A focus on identifying both factors that contribute to ccRCC cell migration and those that are therapeutically targetable is paramount. Methods: We employed several high throughput screening methods to evaluate the pattern of NPTX2 expression in ccRCC samples derived from patient tumor tissues. This included high throughput gene array analysis, evaluation of published gene array datasets, meta-analysis screening, evaluation of patient tissue microarrays (TMA), comparative marker selection and pathway analysis. NPTX2 shRNAs were designed to target and abrogate NPTX2 expression in a cohort of established ccRCC cell lines, and resulting changes in proliferation, viability, and tumor cell migration were evaluated. We also investigated the expression of known NPTX2 binding partners in ccRCC, and evaluated their role in NPTX2 mediated tumorigenic activity. The ionotropic glutamate receptor 4 (GluR4) subunit was identified to be expressed in ccRCC tissue, and we further demonstrate a tumorigenic role for this receptor in ccRCC. • NPTX2 is highly over-expressed in ccRCC at the transcriptional and protein level • NPTX2 expression is specific for the clear cell subtype of RCC • NPTX2 consistently is the most differentially expressed gene transcript in ccRCC • NPTX2 expression is strongly correlated with advanced and metastatic ccRCC • Loss of NPTX2 expression leads to decreased tumor cell growth, loss of tumor cell viability, and decreased invasive capacity • We identify that GluR4 complexes with NPTX2 in ccRCC tumor cells • GluR4 is over-expressed in ccRCC tissue, and demonstrates the highest level of expression in metastatic tissues • Loss of GluR4 expression similarly leads to decreased cell proliferation, loss of tumor cell viability, and decreased invasive capacity of ccRCC cells A) Nextbio was used to assess publicly available expression datasets for NPTX2 in the context of normal vs. tumor, clear cell vs. other subtypes, and localized vs. advanced ccRCC. B) Meta-analysis of gene expression in 13 RCC gene expression datasets for NPTX2. C) IHC for NPTX2 protein expression in patient normal and matched ccRCC tissue across all stages of disease. Stage I, II, III, IV, met: normal n=44, 32, 35, 7, and 6 and tumor n= 41, 26, 33, 10, and 17 respectively. Expression is presented as mean H-score +/- standard deviation. A) mRNA expression of lentiviral NT control and GluR4 targeted (sh1676) ccRCC cell lines. B) Western blot to examine apoptosis via PARP cleavage and C) propidium iodide stain for cell death quantitation in NT and GluR4 targeted ccRCC cells. D) Proliferation assay in ccRCC cells treated with CFM-2, an allosteric AMPA antagonist vs. control. E) Invasion assay evaluating invasive capacity in KIJ265T and A498 (high endogenous NPTX2 and GluR4 expression) treated with CFM-2 vs. control. Invasion is quantitated as number of invading cells per visual field RCC: • Renal cell carcinoma (RCC) is the third most prevalent urological cancer, the 10 th most common cause of cancer death in men and the 9 th most common cause in women. • The clear cell variant of RCC (ccRCC) is the most common subtype accounting for ~80% of all renal cancers. • Due to its asymptomatic nature, it is estimated that up to 30% of patients present with metastatic ccRCC at time of diagnosis. Furthermore, due to its highly metastatic nature 20-30% of patients diagnosed with localized disease will relapse with metastatic ccRCC. • For individuals presenting with advanced disease, treatment options are limited with no current drug therapy leading to long term survival with the exception of 6-7% of patients who respond to interleukin-2. • ccRCC rarely responds to chemotherapy and radiation therapies, and drug resistance develops rapidly with application of targeted therapies. • Genetic factors contributing to ccRCC development, progression, and metastasis are poorly defined. NPTX2: • Neuronal pentraxin 2 (NPTX2) belongs to a class of secreted proteins characterized by their pentraxin domain, and are related to C-reactive protein (CRP). • NPTX2 is typically expressed in nervous system, testicular, pancreatic, skeletal muscle, and hepatic tissues, with little to no expression observed in normal renal tissue. • NPTX2 is best characterized for its role in neurite outgrowth and synaptic plasticity of neuronal cells: mediates the clustering of the AMPA family of ionic glutamate receptors, forming ion permeable channels during excitatory synaptogenesis. A, ccRCC vs normal; B, ccRCC vs chromophobe RCC; C, ccRCC vs granular RCC; D, ccRCC vs papillary RCC A B C D B C A) Western blot of normal and ccRCC cells for NPTX2 expression. B) mRNA expression of lentiviral NT control and NPTX2 targeted (sh1316) ccRCC cell lines. C) Propidium iodide stain for cell death quantitation and D) western blot to examine apoptosis via PARP cleavage of NT and NPTX2 targeted ccRCC cells. E) Invasion assay evaluating invasive capacity in KIJ265T and A498 (high endogenous NPTX2 expression) with loss of NPTX2, and overexpression of NPTX2 in Caki1 and RWV366T (low endogenous NPTX2 expression). Invasion is quantitated as number of invading cells per visual field. F) Immunofluorescence in RWV366T control and NPTX2 overexpression cells and A498 control and NPTX2 knockdown cells for NPTX2 expression and VASP localization- a regulatory protein involved in actin-based motility. A498 NT sh1316 KIJ265T Caki2 NT sh1316 % cell death A498 KIJ265T Caki2 2.7 2.7 1.1 71.3 25.5 25.0 A E F B C Figure 3: GluR4 in ccRCC and Interaction with NPTX2 D E A B • We demonstrate an oncogenic role for NPTX2 for the first time in a cancer system • As NPTX2 is a secreted protein that is consistently and specifically overexpressed in patient ccRCC samples, it presents as: • A candidate diagnostic biomarker for patients with the clear cell subtype of RCC • A candidate prognostic marker for patients at risk of disease recurrence and/or the development of metatstatic disease • An optimal therapeutic target whose inhibition may clinically benefit a broad spectrum of patients FUTURE DIRECTIONS • Development of a monoclonal antibody or small molecule inhibitor of NPTX2 to be tested for therapeutic efficacy in vivo • Evaluate NPTX2 expression in patient blood samples and correlate to disease staging and progression to evaluate efficacy as a diagnostic and prognostic biomarker • Delineate the signaling mechanism of NPTX2 oncogenic activity • Evaluate the efficacy of AMPA antagonists in vivo 1. Kupershmidt I, Su QJ, Grewal A, Sundaresh S, Halperin I, Flynn J, Shekar M, Wang H, Park J, Cui W et al: Ontology-based meta-analysis of global collections of high-throughput public data. PLoS One 2010, 5(9). 2. Ooi A, Wong JC, Petillo D, Roossien D, Perrier-Trudova V, Whitten D, Min BW, Tan MH, Zhang Z, Yang XJ et al: An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma. Cancer Cell 2011, 20(4):511-523. 3. Kort EJ, Farber L, Tretiakova M, Petillo D, Furge KA, Yang XMJ, Cornelius A, Teh BT: The E2F3-Oncomir-1 axis is activated in Wilms' tumor. Cancer Res 2008, 68(11):4034-4038. 4. Wang Y, Roche O, Yan MS, Finak G, Evans AJ, Metcalf JL, Hast BE, Hanna SC, Wondergem B, Furge KA et al: Regulation of endocytosis via the oxygen-sensing pathway. Nat Med 2009, 15(3):319-324. 5. Stickel JS, Weinzierl AO, Hillen N, Drews O, Schuler MM, Hennenlotter J, Wernet D, Muller CA, Stenzl A, Rammensee HG et al: HLA ligand profiles of primary renal cell carcinoma maintained in metastases. Cancer Immunol Immun 2009, 58(9):1407-1417. 6. Yusenko MV, Zubakov D, Kovacs G: Gene expression profiling of chromophobe renal cell carcinomas and renal oncocytomas by Affymetrix GeneChip using pooled and individual tumours. Int J Biol Sci 2009, 5(6):517-527. 7. Jones J, Otu H, Spentzos D, Kolia S, Inan M, Beecken WD, Fellbaum C, Gu XS, Joseph M, Pantuck AJ et al: Gene signatures of progression and metastasis in renal cell cancer. Clin Cancer Res 2005, 11(16):5730-5739. 8. Lenburg ME, Liou LS, Gerry NP, Frampton GM, Cohen HT, Christman MF: Previously unidentified changes in renal cell carcinoma gene expression identified by parametric analysis of microarray data. Bmc Cancer 2003, 3. 9. Gumz ML, Zou H, Kreinest PA, Childs AC, Belmonte LS, LeGrand SN, Wu KJ, Luxon BA, Sinha M, Parker AS et al: Secreted frizzled-related protein 1 loss contributes to tumor phenotype of clear cell renal cell carcinoma. Clin Cancer Res 2007, 13(16):4740- 4749. 10. Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, Leng N, Pavia-Jimenez A, Wang S, Yamasaki T, Zhrebker L, Sivanand S, Spence P et al: BAP1 loss defines a new class of renal cell carcinoma. Nat Genet 2012, 44(7):751-759. 11. The International Genomics Consortium (IGC). The expO project (Expression Project for Oncology) [www.intgen.org] 12. Higgins JPT, Shinghal R, Gill H, Reese JH, Terris M, Cohen RJ, Fero M, Pollack JR, van de Rijn M, Brooks JD: Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray. Am J Pathol 2003, 162(3):925-932. 13. Beleut M, Zimmermann P, Baudis M, Bruni N, Buhlmann P, Laule O, Luu VD, Gruissem W, Schraml P, Moch H: Integrative genome-wide expression profiling identifies three distinct molecular subgroups of renal cell carcinoma with different patient outcome. Bmc Cancer 2012, 12. 14. Tan XJ, Zhai YJ, Chang WJ, Hou JG, He SQ, Lin LP, Yu YW, Xu DF, Xiao JR, Ma LY et al: Global analysis of metastasis-associated gene expression in primary cultures from clinical specimens of clear-cell renal-cell carcinoma. Int J Cancer 2008, 123(5):1080- 1088. 15. Cifola I, Spinelli R, Beltrame L, Peano C, Fasoli E, Ferrero S, Bosari S, Signorini S, Rocco F, Perego R et al: Genome-wide screening of copy number alterations and LOH events in renal cell carcinomas and integration with gene expression profile. Mol Cancer 2008, 7:6. 16. Williams AA, Higgins JP, Zhao H, Ljunberg B, Brooks JD: CD 9 and vimentin distinguish clear cell from chromophobe renal cell carcinoma. BMC Clin Pathol 2009, 9:9. 17. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11-30. 18. Haddad H, Rini BI. Current treatment considerations in metastatic renal cell carcinoma. Curr Treat Options Oncol. 2012;13:212-29. 19. Motzer RJ, Bacik J, Mazumdar M. Prognostic factors for survival of patients with stage IV renal cell carcinoma: memorial sloan-kettering cancer center experience. Clin Cancer Res. 2004;10:6302S-3S. 20. Hsu YC, Perin MS. Human neuronal pentraxin II (NPTX2): conservation, genomic structure, and chromosomal localization. Genomics. 1995;28:220-7. 21. O'Brien R, Xu D, Mi R, Tang X, Hopf C, Worley P. Synaptically targeted narp plays an essential role in the aggregation of AMPA receptors at excitatory synapses in cultured spinal neurons. J Neurosci. 2002;22:4487-98. 22. Sia GM, Beique JC, Rumbaugh G, Cho R, Worley PF, Huganir RL. Interaction of the N-terminal domain of the AMPA receptor GluR4 subunit with the neuronal pentraxin NP1 mediates GluR4 synaptic recruitment. Neuron. 2007;55:87-102. A) mRNA expression profile of GluR1-4 and NPTXR in normal vs. ccRCC cell lines. B) Western blot evaluating GluR4 protein expression in normal and ccRCC cells. C) IHC for GluR4 protein expression in patient normal and matched ccRCC tissue across all stages of disease. Stage I, II, III, IV, met: normal n=45, 35, 38, 8, and 6 and tumor n= 39, 29, 34, 8, and 21 respectively. Expression is presented as mean H-score +/- standard deviation. D) Immunoprecipitation of HA epitope tagged NPTX2 for Flag epitope tagged GluR4 expression, and the reciprocal in 2 ccRCC cell lines. E) Immunofluorescence for NPTX2 expression in non-permeabilized Caki2 NT control, shNPTX2, and shGluR4 cells. Figure 2: Role of NPTX2 in ccRCC Viability and Invasion
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CONCLUSIONS
STUDY DESIGN
BACKGROUND
Neuronal Pentraxin2: A Novel Tumor-Specific Molecular Target That Mediates Clear Cell Renal Cell Carcinoma Malignancy
Christina A. von Roemeling1, Derek Radisky1, Laura Marlow1, Simon J. Cooper1, Stefan K Grebe2, Panagiotis Z. Anastasiadis1, Han W. Tun1,3, John A. Copland1
1Department of Cancer Biology, Mayo Clinic Florida, Jacksonville, Florida; 2Laboratory Medicine and Pathology, Mayo Clinic Minnesota, Rochester, MN;
3Department of Hematology/Oncology, Mayo Clinic Florida, Jacksonville, Florida April 7, 2014: Abstract LB-111
y
CLINICAL IMPACT
RESULTS
REFERENCES
A
Figure 1: NPTX2 Expression Profile in ccRCC
Figure 4: Loss of GluR4 Recapitulates NPTX2 Knockdown Phenotype
D E
C
Translational Relevance: Patients with metastatic ccRCC have poor prognoses, with an estimated 5 year overall survival of less than 10%. This is due to lack of remedial therapies that produce durable disease stabilization or tumor regression. Drug resistance is a hallmark of ccRCC and is linked to cancer cell heterogeneity. Additionally, there is a lack of known molecular factors that can be targeted pharmacologically. The clear cell variant of RCC also frequently manifests as metastatic disease, likely due to its disposition for increased migratory capacity and the formation of undetectable micrometastases. A focus on identifying both factors that contribute to ccRCC cell migration and those that are therapeutically targetable is paramount.
Methods:
We employed several high throughput screening methods to evaluate the pattern of NPTX2 expression in ccRCC samples derived from patient tumor tissues. This included high throughput gene array analysis, evaluation of published gene array datasets, meta-analysis screening, evaluation of patient tissue microarrays (TMA), comparative marker selection and pathway analysis. NPTX2 shRNAs were designed to target and abrogate NPTX2 expression in a cohort of established ccRCC cell lines, and resulting changes in proliferation, viability, and tumor cell migration were evaluated. We also investigated the expression of known NPTX2 binding partners in ccRCC, and evaluated their role in NPTX2 mediated tumorigenic activity. The ionotropic glutamate receptor 4 (GluR4) subunit was identified to be expressed in ccRCC tissue, and we further demonstrate a tumorigenic role for this receptor in ccRCC.
• NPTX2 is highly over-expressed in ccRCC at the transcriptional and protein level • NPTX2 expression is specific for the clear cell subtype of RCC • NPTX2 consistently is the most differentially expressed gene transcript in ccRCC • NPTX2 expression is strongly correlated with advanced and metastatic ccRCC • Loss of NPTX2 expression leads to decreased tumor cell growth, loss of tumor
cell viability, and decreased invasive capacity • We identify that GluR4 complexes with NPTX2 in ccRCC tumor cells • GluR4 is over-expressed in ccRCC tissue, and demonstrates the highest level of
expression in metastatic tissues • Loss of GluR4 expression similarly leads to decreased cell proliferation, loss of
tumor cell viability, and decreased invasive capacity of ccRCC cells
A) Nextbio was used to assess publicly available expression datasets for NPTX2 in the context of normal vs. tumor, clear cell vs. other subtypes, and localized vs. advanced ccRCC. B) Meta-analysis of gene expression in 13 RCC gene expression datasets for NPTX2. C) IHC for NPTX2 protein expression in patient normal and matched ccRCC tissue across all stages of disease. Stage I, II, III, IV, met: normal n=44, 32, 35, 7, and 6 and tumor n= 41, 26, 33, 10, and 17 respectively. Expression is presented as mean H-score +/- standard deviation.
A) mRNA expression of lentiviral NT control and GluR4 targeted (sh1676) ccRCC cell lines. B) Western blot to examine apoptosis via PARP cleavage and C) propidium iodide stain for cell death quantitation in NT and GluR4 targeted ccRCC cells. D) Proliferation assay in ccRCC cells treated with CFM-2, an allosteric AMPA antagonist vs. control. E) Invasion assay evaluating invasive capacity in KIJ265T and A498 (high endogenous NPTX2 and GluR4 expression) treated with CFM-2 vs. control. Invasion is quantitated as number of invading cells per visual field
RCC: • Renal cell carcinoma (RCC) is the third most prevalent urological cancer, the 10th
most common cause of cancer death in men and the 9th most common cause in women.
• The clear cell variant of RCC (ccRCC) is the most common subtype accounting for ~80% of all renal cancers.
• Due to its asymptomatic nature, it is estimated that up to 30% of patients present with metastatic ccRCC at time of diagnosis. Furthermore, due to its highly metastatic nature 20-30% of patients diagnosed with localized disease will relapse with metastatic ccRCC.
• For individuals presenting with advanced disease, treatment options are limited with no current drug therapy leading to long term survival with the exception of 6-7% of patients who respond to interleukin-2.
• ccRCC rarely responds to chemotherapy and radiation therapies, and drug resistance develops rapidly with application of targeted therapies.
• Genetic factors contributing to ccRCC development, progression, and metastasis are poorly defined.
NPTX2: • Neuronal pentraxin 2 (NPTX2) belongs to a class of secreted proteins characterized
by their pentraxin domain, and are related to C-reactive protein (CRP). • NPTX2 is typically expressed in nervous system, testicular, pancreatic, skeletal
muscle, and hepatic tissues, with little to no expression observed in normal renal tissue.
• NPTX2 is best characterized for its role in neurite outgrowth and synaptic plasticity of neuronal cells: mediates the clustering of the AMPA family of ionic glutamate receptors, forming ion permeable channels during excitatory synaptogenesis.
A, ccRCC vs normal; B, ccRCC vs chromophobe RCC; C, ccRCC vs granular RCC; D, ccRCC vs papillary RCC
A
B
C
D
B
C
A) Western blot of normal and ccRCC cells for NPTX2 expression. B) mRNA expression of lentiviral NT control and NPTX2 targeted (sh1316) ccRCC cell lines. C) Propidium iodide stain for cell death quantitation and D) western blot to examine apoptosis via PARP cleavage of NT and NPTX2 targeted ccRCC cells. E) Invasion assay evaluating invasive capacity in KIJ265T and A498 (high endogenous NPTX2 expression) with loss of NPTX2, and overexpression of NPTX2 in Caki1 and RWV366T (low endogenous NPTX2 expression). Invasion is quantitated as number of invading cells per visual field. F) Immunofluorescence in RWV366T control and NPTX2 overexpression cells and A498 control and NPTX2 knockdown cells for NPTX2 expression and VASP localization- a regulatory protein involved in actin-based motility.
A498
NT
sh13
16
KIJ265T Caki2
NT sh1316 % cell death
A498 KIJ265T Caki2
2.7 2.7 1.1
71.3 25.5 25.0
A
E F
B C
Figure 3: GluR4 in ccRCC and Interaction with NPTX2
D E
A B
• We demonstrate an oncogenic role for NPTX2 for the first time in a cancer system
• As NPTX2 is a secreted protein that is consistently and specifically overexpressed in patient ccRCC samples, it presents as: • A candidate diagnostic biomarker for patients with the clear cell subtype of
RCC • A candidate prognostic marker for patients at risk of disease recurrence and/or
the development of metatstatic disease • An optimal therapeutic target whose inhibition may clinically benefit a broad
spectrum of patients
FUTURE DIRECTIONS • Development of a monoclonal antibody or small molecule inhibitor of NPTX2 to
be tested for therapeutic efficacy in vivo • Evaluate NPTX2 expression in patient blood samples and correlate to disease
staging and progression to evaluate efficacy as a diagnostic and prognostic biomarker
• Delineate the signaling mechanism of NPTX2 oncogenic activity • Evaluate the efficacy of AMPA antagonists in vivo
1. Kupershmidt I, Su QJ, Grewal A, Sundaresh S, Halperin I, Flynn J, Shekar M, Wang H, Park J, Cui W et al: Ontology-based meta-analysis of global collections of high-throughput public data. PLoS One 2010, 5(9). 2. Ooi A, Wong JC, Petillo D, Roossien D, Perrier-Trudova V, Whitten D, Min BW, Tan MH, Zhang Z, Yang XJ et al: An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma. Cancer Cell 2011, 20(4):511-523. 3. Kort EJ, Farber L, Tretiakova M, Petillo D, Furge KA, Yang XMJ, Cornelius A, Teh BT: The E2F3-Oncomir-1 axis is activated in Wilms' tumor. Cancer Res 2008, 68(11):4034-4038. 4. Wang Y, Roche O, Yan MS, Finak G, Evans AJ, Metcalf JL, Hast BE, Hanna SC, Wondergem B, Furge KA et al: Regulation of endocytosis via the oxygen-sensing pathway. Nat Med 2009, 15(3):319-324. 5. Stickel JS, Weinzierl AO, Hillen N, Drews O, Schuler MM, Hennenlotter J, Wernet D, Muller CA, Stenzl A, Rammensee HG et al: HLA ligand profiles of primary renal cell carcinoma maintained in metastases. Cancer Immunol Immun 2009, 58(9):1407-1417. 6. Yusenko MV, Zubakov D, Kovacs G: Gene expression profiling of chromophobe renal cell carcinomas and renal oncocytomas by Affymetrix GeneChip using pooled and individual tumours. Int J Biol Sci 2009, 5(6):517-527. 7. Jones J, Otu H, Spentzos D, Kolia S, Inan M, Beecken WD, Fellbaum C, Gu XS, Joseph M, Pantuck AJ et al: Gene signatures of progression and metastasis in renal cell cancer. Clin Cancer Res 2005, 11(16):5730-5739. 8. Lenburg ME, Liou LS, Gerry NP, Frampton GM, Cohen HT, Christman MF: Previously unidentified changes in renal cell carcinoma gene expression identified by parametric analysis of microarray data. Bmc Cancer 2003, 3. 9. Gumz ML, Zou H, Kreinest PA, Childs AC, Belmonte LS, LeGrand SN, Wu KJ, Luxon BA, Sinha M, Parker AS et al: Secreted frizzled-related protein 1 loss contributes to tumor phenotype of clear cell renal cell carcinoma. Clin Cancer Res 2007, 13(16):4740-
4749. 10. Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, Leng N, Pavia-Jimenez A, Wang S, Yamasaki T, Zhrebker L, Sivanand S, Spence P et al: BAP1 loss defines a new class of renal cell carcinoma. Nat Genet 2012, 44(7):751-759. 11. The International Genomics Consortium (IGC). The expO project (Expression Project for Oncology) [www.intgen.org] 12. Higgins JPT, Shinghal R, Gill H, Reese JH, Terris M, Cohen RJ, Fero M, Pollack JR, van de Rijn M, Brooks JD: Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray. Am J Pathol 2003, 162(3):925-932. 13. Beleut M, Zimmermann P, Baudis M, Bruni N, Buhlmann P, Laule O, Luu VD, Gruissem W, Schraml P, Moch H: Integrative genome-wide expression profiling identifies three distinct molecular subgroups of renal cell carcinoma with different patient outcome.
Bmc Cancer 2012, 12. 14. Tan XJ, Zhai YJ, Chang WJ, Hou JG, He SQ, Lin LP, Yu YW, Xu DF, Xiao JR, Ma LY et al: Global analysis of metastasis-associated gene expression in primary cultures from clinical specimens of clear-cell renal-cell carcinoma. Int J Cancer 2008, 123(5):1080-
1088. 15. Cifola I, Spinelli R, Beltrame L, Peano C, Fasoli E, Ferrero S, Bosari S, Signorini S, Rocco F, Perego R et al: Genome-wide screening of copy number alterations and LOH events in renal cell carcinomas and integration with gene expression profile. Mol Cancer
2008, 7:6. 16. Williams AA, Higgins JP, Zhao H, Ljunberg B, Brooks JD: CD 9 and vimentin distinguish clear cell from chromophobe renal cell carcinoma. BMC Clin Pathol 2009, 9:9. 17. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin. 2013;63:11-30. 18. Haddad H, Rini BI. Current treatment considerations in metastatic renal cell carcinoma. Curr Treat Options Oncol. 2012;13:212-29. 19. Motzer RJ, Bacik J, Mazumdar M. Prognostic factors for survival of patients with stage IV renal cell carcinoma: memorial sloan-kettering cancer center experience. Clin Cancer Res. 2004;10:6302S-3S. 20. Hsu YC, Perin MS. Human neuronal pentraxin II (NPTX2): conservation, genomic structure, and chromosomal localization. Genomics. 1995;28:220-7. 21. O'Brien R, Xu D, Mi R, Tang X, Hopf C, Worley P. Synaptically targeted narp plays an essential role in the aggregation of AMPA receptors at excitatory synapses in cultured spinal neurons. J Neurosci. 2002;22:4487-98. 22. Sia GM, Beique JC, Rumbaugh G, Cho R, Worley PF, Huganir RL. Interaction of the N-terminal domain of the AMPA receptor GluR4 subunit with the neuronal pentraxin NP1 mediates GluR4 synaptic recruitment. Neuron. 2007;55:87-102.
A) mRNA expression profile of GluR1-4 and NPTXR in normal vs. ccRCC cell lines. B) Western blot evaluating GluR4 protein expression in normal and ccRCC cells. C) IHC for GluR4 protein expression in patient normal and matched ccRCC tissue across all stages of disease. Stage I, II, III, IV, met: normal n=45, 35, 38, 8, and 6 and tumor n= 39, 29, 34, 8, and 21 respectively. Expression is presented as mean H-score +/- standard deviation. D) Immunoprecipitation of HA epitope tagged NPTX2 for Flag epitope tagged GluR4 expression, and the reciprocal in 2 ccRCC cell lines. E) Immunofluorescence for NPTX2 expression in non-permeabilized Caki2 NT control, shNPTX2, and shGluR4 cells.
Figure 2: Role of NPTX2 in ccRCC Viability and Invasion
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