Human papillomavirus (HPV) is the most prevalent sexually transmitted infection and a necessary cause of cervical cancer, the third most common cancer in women worldwide [
1]. More than 99% of cervical cancers contain DNA of HPV high-risk types [
2]. In addition, HPV DNA is also found in a significant percentage of other anogenital lesions as well as oral and oropharyngeal tumors [
3‐
5]. HPV-16 is the most prevalent type and it is found in almost 50% of all cervical cancer cases [
6]. The two major HPV oncoproteins, E6 and E7, are consistently expressed in all HPV positive cancers. The expression of these proteins in primary human keratinocytes effectively induces their immortalization [
7]. Furthermore, when grown under conditions that allow stratification and the formation of skin-like structures, cells immortalized with E6 and E7 from high-risk HPV types display morphological hallmarks of high-grade squamous intra-epithelial lesions, well-established precursors of cervical cancer [
8]. The best characterized activity of HPV-16 E6 and E7 is their ability to bind to and induce the proteasome-mediated degradation of tumor suppressors p53 and pRb, respectively [
9,
10]. Besides, both HPV-16 E6 and E7 are able to bind to and alter the biological function of several other cellular proteins [
11]. Among them are many members of the phosphatidylinositol (PI)-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway [
12]. The PI3K/Akt/mTOR signaling axis plays a very important role in HPV-induced carcinogenesis by acting through multiple cellular and molecular events [
13]. Although the regulation of mTOR through PI3K/AKT has been extensively described, another mechanism contributing to activation of mTOR has been proposed. Phosphatidic acid (PA), a product of phospholipase D, is required for mTORC1 activation by mitogens as well as amino acid signals [
14‐
16]. More recently, PA was identified as a major product capable of displacing DEPTOR, a mTOR binding protein that normally functions to inhibit both mTORC1 and mTORC2 pathways [
17]. PA species with unsaturated fatty acids chains, such as those produced by PLD, bind with high affinity to the FRB domain of mTOR in a manner that is competitive with its inhibitor rapamycin. As a consequence, elevated PLD activity has been associated to rapamycin resistance [
18,
19]. In addition, phospholipase D enzymes play a fundamental role in cells: they maintain the integrity of cellular membranes and they participate in cell signaling including cytoskeletal dynamics, cell migration, intracellular protein trafficking, and cell proliferation. Consistent with this data, increased PLD activity has been reported in a large number of human cancers, including breast, colon, gastric, and kidney [
20].
Our results show that upon HPV-16 E7 expression, primary human foreskin keratinocytes upregulate PLD protein levels and activity. Such effect is dependent on the integrity of the E7 LxCxE binding motif and, ultimately to the ability of HPV-16 E7 to induce pRb degradation and promote immortalization. We also show that organotypic cultures of keratinocytes expressing HPV-16 E7 become resistant to the antiproliferative effect of rapamycin.