Falling from grace: HPRT is not suitable as an endogenous control for cancer-related studies

Abstract

HPRT is a housekeeping enzyme involved in recycling guanine and inosine in the purine salvage pathway. As a housekeeping gene, HPRT has been widely used as an endogenous control for molecular studies evaluating changes in gene expression. Yet, recent evidence has shown that HPRT exhibits high variability within malignant samples. We designed this study to determine whether this observed upregulation is consistently found, therefore rendering hprt an unsuitable normalization control in cancer. Utilizing protein and RNA-seq expression, we found that malignant and normal patient samples vary significantly both within the same tissue type and across organ sites. Upon staining for HPRT via immunohistochemistry, we found that expression is highly variable in malignant samples (Lung; Breast; Colon; Prostate; Pancreas;. Similarly, we observed high variability across cell lines via western blotting (p < 0.0001) which was further confirmed using RNA sequencing. Comparing normal and malignant patient samples, we observed consistent upregulation of HPRT expression within malignant samples relative to normal samples (p = 0.0001). These data indicate that HPRT is unsuitable as an endogenous control for cancer-related studies because its expression is highly variable and exceeds that of an appropriate control; therefore, we recommend its discontinued use as a normalization gene. ARTICLE HISTORY

Hypoxanthine Phosphoribosyl Transferase 1 Is Upregulated, Predicts Clinical Outcome and Controls Gene Expression in Breast Cancer

“…HPRT1 is considered a housekeeping gene and is widely used as an endogenous control in gene expression studies; however, recent studies, including our data, have revealed its potential involvement in cancer [ 3 , 4 , 5 ]. Unchecked and aberrant exponential cell growth, a hallmark of cancer, results in increased transcriptional output related to cancer genes.…”

“…Although widely considered to be a housekeeping gene, recent studies have shown HPRT1 expression levels to be highly variable between malignant and normal tissue, suggesting that it is unreliable as an endogenous control in cancer-related studies [ 3 ], and also indicating its plausible role in cancer biology. HPRT1 expression levels varied widely in a large cohort of both normal and malignant samples of different tissue origin, with breast cancer exhibiting the highest average HPRT1 compared to other malignancies [ 4 ]. Studies from various cancers, such as colorectal cancer, have shown elevated levels of HPRT1 in tumor samples compared to normal tissues [ 3 , 4 , 5 ].…”

“…HPRT1 expression levels varied widely in a large cohort of both normal and malignant samples of different tissue origin, with breast cancer exhibiting the highest average HPRT1 compared to other malignancies [ 4 ]. Studies from various cancers, such as colorectal cancer, have shown elevated levels of HPRT1 in tumor samples compared to normal tissues [ 3 , 4 , 5 ]. Additionally, the same study found HPRT1 localizes to the surface of the cell membrane in more progressive types of colorectal cancer [ 4 , 5 ].…”

“…Studies from various cancers, such as colorectal cancer, have shown elevated levels of HPRT1 in tumor samples compared to normal tissues [ 3 , 4 , 5 ]. Additionally, the same study found HPRT1 localizes to the surface of the cell membrane in more progressive types of colorectal cancer [ 4 , 5 ]. Likewise, prostate cancer patient samples showed an increased expression of HPRT1 when compared to normal tissues [ 3 ].…”

IMPDH2 and HPRT expression and a prognostic significance in preoperative and postoperative patients with osteosarcoma

“…Hypoxanthine guanine phosphoribosyltransferase (HPRT) is a key enzyme in salvage pathway responsible for the formation of GMP and IMP by recycling guanine and inosine from degraded DNA and RNA, respectively 26 . To maintain cellular GTP level in somatic cells, HPRT is reliably expressed in low level 27 . Unlike in normal cells, recent study showed that HPRT expression is highly variable in various cancer tissues including lung, breast, colon, prostate and pancreas 27 .…”