Am J Cancer Res 2013;3(2):230-239
Original Article
Correlation of Notch1, pAKT and nuclear NF-κB expression in triple negative
breast cancer
He Zhu, Feriyl Bhaijee, Nivin Ishaq, Dominique J Pepper, Kandis Backus, Alexandra S Brown, Xinchun Zhou, Lucio Miele
Cancer Institute, Department of Pathology, Department of Medicine, Department of Pharmacology and Toxicology, University of
Mississippi Medical Center, Jackson, MS 39216
Received February 8, 2013; Accepted March 15, 2013; Epub April 3, 2013; Published April 13, 2013
Abstract: Gene expression profiling reveals elevated Notch1 mRNA expression in triple negative breast cancers (TNBC), both
basaloid and claudin-low subtypes. Notch ligands, Jagged1 and Jagged2, have been correlated with poor prognosis in TNBC.
AKT, an oncogenic protein kinase family that is activated downstream of Notch in breast cancer cell lines, is frequently activated
in breast cancer. Recent publications suggest that inhibition of cell growth, migration, invasion, and induction of apoptosis
caused by Notch1 or Jagged1 inhibition may be attributed in part to inactivation of the AKT signaling pathway. There is significant
evidence that Notch1 activates NF-κB in several models, and that AKT can mediate NF-κB activation. In this study, we evaluated
Notch1 protein expression by immunohistochemistry (IHC) and correlated this with expression of pAKT and nuclear NF-κB p65
(RelA) in TNBC. A tissue microarray (TMA) containing 32 formalin-fixed, paraf-fin-embedded (FFPE) TNBC tumor specimens was
constructed from the archival tissue database of the Department of Pathology at UMMC and IHC for Notch1 protein, pAKT 1/2/3
(Ser473), and NF-κB, p65 subunit was performed on the TMA with appropriate positive and negative controls. Of the 32 TNBC in
our cohort, 100% expressed Notch1 protein by IHC: 24 (75%) showed cytoplasmic expression, 25 (78%) showed membranous
expression, and 17 (53%) showed both cytoplasmic and membranous expression. Overall, 29 (91%) expressed pAKT by IHC:
28 (97%) showed cytoplasmic expression, 14 (48%) showed nuclear expression and 13 (45%) showed both cytoplasmic and
nuclear expression. Nuclear staining for NF-κB p65 was detected in all 32 TNBC specimens with variable intensities. On
bivariate analysis, cytoplasmic Notch1 was significantly correlated with cytoplasmic pAKT (r = 0.373, P = 0.035) and nuclear NF-
κB (r = 0.483, P = 0.005); both cytoplasmic and nuclear pAKT significantly correlated with nuclear NF-κB (r = 0.391, P = 0.027; r =
0.525, P = 0.002, respectively). These results suggest that 1) the cross-talk between Notch1, AKT and NF-κB identified in
preclinical models may operate in a significant fraction of human TNBC, and 2) combination therapy with agents targeting these
pathways warrants further investigation. (AJCR0000176)
Keywords: Triple negative breast cancers (TNBC), Notch1, AKT, NF-κB, immunohistochemistry (IHC), tissue microarray (TMA)
Address correspondence to: Dr. Lucio Miele, Cancer Institute, University of Mississippi Medical Center, 2500 N. State St., Suite
G751-5, Jackson, MS 39216, USA. Tel: 601-815-6802/6803; Fax: 601-815-6806; E-mail: lmiele@umc.edu
Original Article
Correlation of Notch1, pAKT and nuclear NF-κB expression in triple negative
breast cancer
He Zhu, Feriyl Bhaijee, Nivin Ishaq, Dominique J Pepper, Kandis Backus, Alexandra S Brown, Xinchun Zhou, Lucio Miele
Cancer Institute, Department of Pathology, Department of Medicine, Department of Pharmacology and Toxicology, University of
Mississippi Medical Center, Jackson, MS 39216
Received February 8, 2013; Accepted March 15, 2013; Epub April 3, 2013; Published April 13, 2013
Abstract: Gene expression profiling reveals elevated Notch1 mRNA expression in triple negative breast cancers (TNBC), both
basaloid and claudin-low subtypes. Notch ligands, Jagged1 and Jagged2, have been correlated with poor prognosis in TNBC.
AKT, an oncogenic protein kinase family that is activated downstream of Notch in breast cancer cell lines, is frequently activated
in breast cancer. Recent publications suggest that inhibition of cell growth, migration, invasion, and induction of apoptosis
caused by Notch1 or Jagged1 inhibition may be attributed in part to inactivation of the AKT signaling pathway. There is significant
evidence that Notch1 activates NF-κB in several models, and that AKT can mediate NF-κB activation. In this study, we evaluated
Notch1 protein expression by immunohistochemistry (IHC) and correlated this with expression of pAKT and nuclear NF-κB p65
(RelA) in TNBC. A tissue microarray (TMA) containing 32 formalin-fixed, paraf-fin-embedded (FFPE) TNBC tumor specimens was
constructed from the archival tissue database of the Department of Pathology at UMMC and IHC for Notch1 protein, pAKT 1/2/3
(Ser473), and NF-κB, p65 subunit was performed on the TMA with appropriate positive and negative controls. Of the 32 TNBC in
our cohort, 100% expressed Notch1 protein by IHC: 24 (75%) showed cytoplasmic expression, 25 (78%) showed membranous
expression, and 17 (53%) showed both cytoplasmic and membranous expression. Overall, 29 (91%) expressed pAKT by IHC:
28 (97%) showed cytoplasmic expression, 14 (48%) showed nuclear expression and 13 (45%) showed both cytoplasmic and
nuclear expression. Nuclear staining for NF-κB p65 was detected in all 32 TNBC specimens with variable intensities. On
bivariate analysis, cytoplasmic Notch1 was significantly correlated with cytoplasmic pAKT (r = 0.373, P = 0.035) and nuclear NF-
κB (r = 0.483, P = 0.005); both cytoplasmic and nuclear pAKT significantly correlated with nuclear NF-κB (r = 0.391, P = 0.027; r =
0.525, P = 0.002, respectively). These results suggest that 1) the cross-talk between Notch1, AKT and NF-κB identified in
preclinical models may operate in a significant fraction of human TNBC, and 2) combination therapy with agents targeting these
pathways warrants further investigation. (AJCR0000176)
Keywords: Triple negative breast cancers (TNBC), Notch1, AKT, NF-κB, immunohistochemistry (IHC), tissue microarray (TMA)
Address correspondence to: Dr. Lucio Miele, Cancer Institute, University of Mississippi Medical Center, 2500 N. State St., Suite
G751-5, Jackson, MS 39216, USA. Tel: 601-815-6802/6803; Fax: 601-815-6806; E-mail: lmiele@umc.edu
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American Journal of Cancer Research
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