A Combined Gene Signature of Hypoxia and Notch Pathway in Human Glioblastoma and Its Prognostic Relevance

Abstract

Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor—clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures.

FAT1 modulates EMT and stemness genes expression in hypoxic glioblastoma

“…FAT1 expression positively correlates with the expression of hypoxia, EMT and stemness markers in GBM tumors : mRNA expression of EMT (Snail/LOX/Vimentin/N‐cad/E‐cad) and stemness (SOX2/OCT4/Nestin) markers was analyzed in a cohort of GBM samples ( n = 31) previously analyzed for FAT1 and hypoxia markers (PGK1/VEGF/CA9/HIF‐1α) . FAT1 was upregulated (≥1.5‐fold of normal brain RNA) in 45% of the tumors while hypoxia markers were upregulated in ≥55% of the tumors [PGK1 (81%), VEGF (77%), CA9 (74%) and HIF‐1α (55%)] (Table S1).…”

“…FAT1 expression positively correlates with the expression of hypoxia, EMT and stemness markers in GBM tumors: mRNA expression of EMT (Snail/LOX/Vimentin/N-cad/E-cad) and stemness (SOX2/OCT4/Nestin) markers was analyzed in a cohort of GBM samples (n 5 31) previously analyzed for FAT1 and hypoxia markers (PGK1/VEGF/CA9/HIF-1a). 15,19 FAT1 was upregulated (1.5-fold of normal brain RNA) in 45% of the tumors while hypoxia markers were upregulated in 55% of the tumors [PGK1 (81%), VEGF (77%), CA9 (74%) and HIF-1a (55%)] (Table S1). On analyzing the same GBM tumors for EMT and stemness markers, we found increased expression (1.5-fold) of EMT markers [LOX (61%), Vimentin (VIM) (55%), Snail (42%) and N-cad (26%)] in 26% of the tumors and stemness markers [SOX2 (84%), Nestin (81%) and OCT4 (39%)] in 39% of the tumors (Table S1).…”

“…We also showed that FAT1 upregulates HIF‐1α expression and functions under hypoxic conditions, thus affecting their metabolic status and invasive properties . Studies have demonstrated that the hypoxic microenvironment with increased HIF‐1α promotes stemness and invasiveness, which favors tumor growth and therapeutic resistance . Here, for the first time, we have demonstrated the role of FAT1 in regulating EMT and stemness properties in GBM under hypoxia, highlighting the importance of FAT1 signaling in aggressive GBM.…”

“…Thirty‐one surgically resected GBM samples were collected from Department of Neurosurgery/Neuropathology, AIIMS, New Delhi, after obtaining ethical clearance (IESC/T‐106/04.03.2011) and written consent from the patients . Control normal human‐brain RNA was purchased from Stratagene/Clontech (La Jolla/Mountain View, CA).…”

Biomarkers in Glioblastoma multiforme. Evidences from a literature review

“…Details on 11 studies (445 patients) analyzing the prognostic/predictive value of VEGF expression are given in Table 3 [8,14,[46][47][48][49][50][51][52][53][54]. VEGF expression was mostly analyzed by immunohistochemistry (7/11 studies, 63.6%).…”