ABSTRACT
BACKGROUND: The aim of this study is to determine whether Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) could be used as a biomarker for the diagnosis and treatment of B cell malignancies. With 4.3% of all new cancers diagnosed as Non-Hodgkin lymphoma, finding new biomarkers for the treatment of B cell cancers is an ongoing pursuit. HPRT is a nucleotide salvage pathway enzyme responsible for the synthesis of guanine and inosine throughout the cell cycle. METHODS: Raji cells were used for this analysis due to their high HPRT internal expression. Internal expression was evaluated utilizing western blotting and RNA sequencing. Surface localization was analyzed using flow cytometry, confocal microscopy, and membrane biotinylation. To determine the source of HPRT surface expression, a CRISPR knockdown of HPRT was generated and confirmed using western blotting. To determine clinical significance, patient blood samples were collected and analyzed for HPRT surface localization. RESULTS: We found surface localization of HPRT on both Raji cancer cells and in 77% of the malignant ALL samples analyzed and observed no significant expression in healthy cells. Surface expression was confirmed in Raji cells with confocal microscopy, where a direct overlap between HPRT specific antibodies and a membrane-specific dye was observed. HPRT was also detected in biotinylated membranes of Raji cells. Upon HPRT knockdown in Raji cells, we found a significant reduction in surface expression, which shows that the HPRT found on the surface originates from the cells themselves. Finally, we found that cells that had elevated levels of HPRT had a direct correlation to XRCC2, BRCA1, PIK3CA, MSH2, MSH6, WDYHV1, AK7, and BLMH expression and an inverse correlation to PRKD2, PTGS2, TCF7L2, CDH1, IL6R, MC1R, AMPD1, TLR6, and BAK1 expression. Of the 17 genes with significant correlation, 9 are involved in cellular proliferation and DNA synthesis, regulation, and repair. CONCLUSIONS: As a surface biomarker that is found on malignant cells and not on healthy cells, HPRT could be used as a surface antigen for targeted immunotherapy. In addition, the gene correlations show that HPRT may have an additional role in regulation of cancer proliferation that has not been previously discovered.
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; 89.2-111.8, Breast; 66.7-98.3, Colon; 85.3-129.7, Prostate; 90.8-155.4, Pancreas; 74.1-132.1). 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.
ABSTRACT
BACKGROUND: Incidence of endometrial cancer are rising both in the United States and worldwide. As endometrial cancer becomes more prominent, the need to develop and characterize biomarkers for early stage diagnosis and the treatment of endometrial cancer has become an important priority. Several biomarkers currently used to diagnose endometrial cancer are directly related to obesity. Although epigenetic and mutational biomarkers have been identified and have resulted in treatment options for patients with specific aberrations, many tumors do not harbor those specific aberrations. A promising alternative is to determine biomarkers based on differential gene expression, which can be used to estimate prognosis. METHODS: We evaluated 589 patients to determine differential expression between normal and malignant patient samples. We then supplemented these evaluations with immunohistochemistry staining of endometrial tumors and normal tissues. Additionally, we used the Library of Integrated Network-based Cellular Signatures to evaluate the effects of 1826 chemotherapy drugs on 26 cell lines to determine the effects of each drug on HPRT1 and AURKA expression. RESULTS: Expression of HPRT1, Jag2, AURKA, and PGK1 were elevated when compared to normal samples, and HPRT1 and PGK1 showed a stepwise elevation in expression that was significantly related to cancer grade. To determine the prognostic potential of these genes, we evaluated patient outcome and found that levels of both HPRT1 and AURKA were significantly correlated with overall patient survival. When evaluating drugs that had the most significant effect on lowering the expression of HPRT1 and AURKA, we found that Topo I and MEK inhibitors were most effective at reducing HPRT1 expression. Meanwhile, drugs that were effective at reducing AURKA expression were more diverse (MEK, Topo I, MELK, HDAC, etc.). The effects of these drugs on the expression of HPRT1 and AURKA provides insight into their role within cellular maintenance. CONCLUSIONS: Collectively, these data show that JAG2, AURKA, PGK1, and HRPT1 have the potential to be used independently as diagnostic, prognostic, or treatment biomarkers in endometrial cancer. Expression levels of these genes may provide physicians with insight into tumor aggressiveness and chemotherapy drugs that are well suited to individual patients.
