The HPV16 E6 and E7 oncogenes modify Glutamine metabolism in Cervical Cancers

Abstract

Metabolic reprogramming is an established hallmark of cancer. Malignant cells induce metabolic adjustments to meet their high energy demands and sustain rapid proliferation. These metabolic changes involve modifications in glutamine metabolism including upregulation of transporters and enzymes, and regulation of glutamine uptake by oncogenes and tumor suppressors, to promote glutaminolysis in cancer cells. Cervical cancer continues to pose a significant health burden for women worldwide. Persistent infection with a high-risk Human Papillomavirus (HR-HPV) strain, and subsequent upregulation of the viral oncogenes, E6 and E7, constitute the primary risk factor for the development of cervical cancer. Accumulating evidence supports that the viral oncogenes potentially favor metabolic reprogramming; however, the underlying mechanisms remain unknown. The aim of this research was to investigate whether the oncogenes of HPV16, E6 and E7, promote the rewiring of glutamine metabolism in cervical cancer. To address this, we used the in vitro tumor sphere formation assay to assess whether the sphere-forming capabilities of HPV (+) and HPV (-) cervical cancer cells are influenced when glutamine metabolism is hindered or restrained. We found that cervical cancer cells exhibit an impaired ability to form tumor spheres in the absence of glutamine, regardless of HPV presence. Furthermore, transcriptional analysis performed to examine the expression levels of glutamine metabolism-related genes, demonstrated significantly altered expression between HPV (+) and HPV (-) cervical cancer cells. Nevertheless, since we did not identify a consistent expression pattern, we developed a cellular system, where we transduced the HPV (-) C33A cells, to induce expression of E6 and E7, either alone or together, in order to interrogate whether the viral oncogenes are responsible for the differences observed. Transcriptional analysis of C33A-transduced cells revealed that the presence of the E7 oncogene upregulates key genes involved in glutamine metabolism, including the glutamine transporters Slc1a5 and Slc7a5, as well as glutamine metabolism-related enzymes, such as GLS and Glud1, raising the possibility of facilitated glutamine uptake and increased glutaminolysis in HPV-associated cervical cancers. The consumption of glutamine and glutamate was assessed in oncogene-expressing cells, showing that extracellular glutamine was increased, while extracellular glutamate levels were decreased in the presence of the viral oncogenes, suggesting that E6 and E7, affect the consumption of these metabolites. Moreover, we demonstrated that the E7-expressing cells are less sensitive to effects of glutamine starvation in the clonogenic potential of cervical cancer cells, since the presence of E7, not only led to an increase in the number of tumor spheres, but also contributed to the growth of larger tumor spheres. Collectively, our data demonstrate the impact of the HPV16 oncogenes, E6 and E7, in promoting metabolic reprogramming through modifying glutamine metabolism to fulfil the high energy requirements of cervical cancer cells, and subsequently support the cancerous phenotype.