Human Papillomaviruses-Associated Cancers: An Update of Current Knowledge

Abstract

Human papillomaviruses (HPVs), which are small, double-stranded, circular DNA viruses infecting human epithelial cells, are associated with various benign and malignant lesions of mucosa and skin. Intensive research on the oncogenic potential of HPVs started in the 1970s and spread across Europe, including Croatia, and worldwide. Nowadays, the causative role of a subset of oncogenic or high-risk (HR) HPV types, led by HPV-16 and HPV-18, of different anogenital and head and neck cancers is well accepted. Two major viral oncoproteins, E6 and E7, are directly involved in the development of HPV-related malignancies by targeting synergistically various cellular pathways involved in the regulation of cell cycle control, apoptosis, and cell polarity control networks as well as host immune response. This review is aimed at describing the key elements in HPV-related carcinogenesis and the advances in cancer prevention with reference to past and on-going research in Croatia.

Keywords: biomarkers; cancer; epigenetics; human papillomaviruses (HPV); immunology; oncoproteins; proteomics; therapeutics.

Figure 1

Schematic representation of HPV-16 genome. The episomal (circular) form of the viral genome comprises 7904 bp coding for a limited number of regulatory (E, early) and structural (L, late) viral proteins. LCR (long control region) is a noncoding region that regulates transcription of the viral genome. E1 codes for an ATPase (DNA-dependent adenosine triphosphatase) and DNA helicase, which serves to initiate DNA replication jointly with the E2 protein. In addition to starting replication, E2 acts as a transcriptional regulator together with the E2 response elements in the LCR. E4 serves to disrupt the keratin filaments in the upper layers of the epithelium, while E5 induces cell proliferation, angiogenesis, and apoptosis by regulating growth signalling pathways in addition to helping immune evasion. E6 oncoprotein promotes apoptosis evasion and cell-cycle deregulation. E7 promotes uncontrolled proliferation and, together with E6, induces malignant transformation. L1 and L2 genes code for viral capsid proteins, which are expressed only in the late phase of the viral life cycle. HPV DNA integration into a host cell genome is random event although it often occurs close to common fragile sites. Upon integration, most of the regulatory genes (E1, E2, E4, and E5) and the capsid genes (L1 and L2) are lost, but two main oncogenes, E6 and E7, remain uncontrollably expressed and are no longer under negative control of the E2 protein. This gives the cells integrated HPV genomes and a selective growth advantage and promotes the development of cancer.

Figure 2

From HPV infection to malignant development. (A) HPV virions infect cells in the basal layer of the cervical epithelium through microtrauma. The expression of individual genes follows the differentiation of epithelial cells from the basal layer to the squamous cell layers of the epidermis with virion release from the upper layers. In the initial stage of infection, early genes are expressed, and they regulate viral replication and transcription. In persistent infection, cells in the parabasal layer become dysplastic, but the productive viral life cycle continues, with the area of cell proliferation becoming wider along with the progression of cervical intraepithelial lesions (CIN) from CIN1 to CIN2. If the host’s immune system does not resolve the viral infection, and it persists for a long period of time (several years), this can lead to pre-cancerous lesions and then to cancer development. When dysplastic cells encompass the entire thickness of the epithelia (CIN3), the productive viral life cycle is present only in the narrow area at the surface if at all. With malignant transformation, productive viral life cycle becomes abortive (green triangle—E6, E7 expression; blue triangle—E1, E2, E4, E5 expression; pink triangle—L1, L2 expression). (B) The expression level of viral oncoproteins E6 and E7 during HPV carcinogenesis varies as shown. The viral oncoproteins E6 and E7 mainly act on the tumour suppressors p53 and pRb, respectively. E6 binds and degrades p53 via ubiquitin-ligase E6AP, while oncoprotein E7 binds and degrades pRb via cullin-2 ubiquitin ligase complex. In the long term, infection with HR HPV types leads to uncontrolled cellular division (proliferation) without the possibility of repairing genetic defects leading to genetic instability, cell immortalization, and loss of cellular polarity, resulting finally with malignant transformation of host cells.

Figure 3

E6 and E7 oncoproteins of HPV-16. (A) Schematic representation of HPV-16 E6 oncoprotein amino acid sequence and motifs crucial for E6 protein integrity and activity. HPV-16 E6 contains two zinc finger domains that are involved in a subset of interactions with cellular substrates. Some of the most characterized interacting partners are depicted. (B) Schematic representation of HPV-16 E7 oncoprotein amino acid sequence and motifs essential for the oncoprotein stability and function. Three conserved regions of E7 are indicated (CR1-CR3) along with some of their most well-researched interacting partners as well as serines, which are subject to casin kinase II (CKII) phosphorylation, and the zinc-finger region with its interacting partners (adapted from Tomaic, 2016).

Figure 4

HPV E6 and E7 employ a multifaceted approach in modulating cell behaviour. The oncoproteins utilize various strategies and interact with numerous host cellular proteins to modulate various cellular processes important for productive viral life cycle and malignant progression. By the joint actions of E6 and E7, HPV can evade the host immune response and establish a persistent infection and to deregulate programmed cell death pathways, such as apoptosis and anoikis, in order to make the cells more tolerant to the changes in cell polarity and uncontrolled proliferation, which inevitably leads to cell transformation and immortalisation. Concomitantly, E6 and E7 regulate differential expression of micro RNAs in HPV-infected keratinocytes or HPV-induced malignancies. Likewise, the epigenetic signatures of various genes have been found to differentiate between lesions and normal tissue due to the interactions of oncoproteins with methyl- and acetyl-transferases.

Figure 5

The immune microenvironment of HPV-associated cancers. HPV-infected cells express less MHC-I receptors and E-cadherin (E-cad) and, as a consequence, attract and retain less APCs and activate less CD8+ cells. Tumour cells secrete various cytokines and chemokines; IL-6 inhibits DC maturation and contributes to monocyte maturation into TAMs, as does CCL2, while immature DCs secrete the pro-tumorigenic MMP9 under the influence of IL-6. IDO secreted by tumour cells and NKT cells inhibits the cytotoxic activity of NK cells. Naive CD4+ cells mature into T helper and T regulatory cells in the tumour environment. Th2 cells secrete cytokines, which induce monocyte maturation into TAM, whereas TAMs produce the same interleukins that also promote CD4+ differentiation into Th2, creating a feedback loop. TAMs also promote the recruitment of immune-suppressive Tregs with CCL22 and the maturation of CD4+ cells into Tregs with IL-10. Th2, Tregs, and Th17 secrete IL-6, IL-8, IL-10, and IL-17, which are all pro-tumorigenic in the context of HPV-associated tumours, as they inhibit the maturation of DCs and the activation of NK cells. Abbreviations: APC, antigen-presenting cell; CA, cancer cell (HPV induced); CCL, C-C motif chemokine ligand; DC, dendritic cell; MMP9, matrix metallopeptidase 9; E-cad, E-cadherin (calcium-dependent adhesion); IDO, indoleamine 2,3-dioxygenase; IFN, interferon; IL, interleukin; NK(T), natural killer (T) cell; TAM, tumour associated macrophages; Th, helper T cell; Treg, regulatory T cell.

Figure 6

Schematic representation of DNA methylation in HPV-associated cancer. In healthy tissue, DNA methylation occurs often on most cellular gene promoters, resulting in the absence of their expression. In cancer, some relevant genes may become methylated, while most remain unmethylated. Thus, aberrant DNA methylation profile in cancer compared to normal tissue can represent a biomarker for cancer occurrence and even association to HPV infection causing that cancer. Epigenetic drugs, such as demethylating agents (DNA methyltransferase (DNMT) inhibitors), could be used to induce the expression of specific genes that would slow the progression of the cancer and enable restoration of normal phenotype. Hypermethylated genes are presented in orange and hypomethylated in blue.