The Cancer Stem Cell Marker CD133 is Regulated by HIF-1a

Student Researcher:
Katherine Jensen

Supervisors / Principle Investigators:
Dr. Samuel Weiss
Dr. H. Artee Luchman

Additional Authors:
Ian Restall

MD Class of 2021

ABSTRACT

Glioblastoma (GBM), characterized by an aggressive clinical course and striking molecular heterogeneity, remains an incurable brain tumour. Brain tumour stem cells (BTSCs) are a subset of GBM cells that are thought to be responsible for cancer initiation, therapeutic resistance, and disease recurrence. BTSCs display cancer stem cell (CSC) properties of self-renewal, multipotent lineage differentiation, and tumorigenicity. CD133 is one of the most commonly used markers to selectively isolate and study BTSCs as well as CSCs derived from other tumour types.

There is contradicting literature as to whether CD133 can be used as a reliable marker of CSCs. It has been shown that altered CD133 expression in vitro does not necessarily correlate with the associated changes in CSC properties. More recently, studies have suggested that CD133 may be regulated by hypoxia and that BTSCs are enriched in the hypoxic regions of GBM tumours. For this study, BTSCs were used to elucidate the usefulness of CD133 as a CSC marker in GBM. We hypothesized that CD133 is a reflection of hypoxia and hypoxia-inducible factor 1 alpha (HIF1-a) activity.

Rotenone, an electron transport chain inhibitor, was used to inhibit HIF1-a activity; whereas, DMOG, a cell permeable prolyl-4-hydroxylase inhibitor, was used to increase HIF1-a levels. To study the effects of modulating HIF1-a activity in BTSCs, qPCR was used to identify changes in gene expression, western blotting was used to look at protein levels, and flow cytometry was used to look at changes in the percentage of CD133+ cells. With DMOG treatment, we observed an increase in RNA and protein expression of genes downstream of HIF1-a, including CD133, carbonic anhydrase IX (CA9), hexokinase II (HKII), BNIP3, pyruvate dehydrogenase kinase 1 (PDK1), and glucose transporter type 1 (GLUT1); whereas, with rotenone, there was a significant decrease. We further observed that HIF1-a activity correlated with the percentage of CD133+ cells.

This work will further our understanding of the enigmatic role of CD133 in BTSCs. We aim to demonstrate that CD133 is a readout of hypoxia and a poor marker of CSCs in vitro as it does not reflect changes in stemness; however, it remains a relevant marker in tissue.

 
 

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