Bioinformatics analysis of immune characteristics in tumors with alternative carcinogenesis pathways induced by human papillomaviruses

Human papillomaviruses (HPVs) are associated with approximately 4% of human cancers worldwide [1]. They are involved in nearly all cases of cervical carcinoma (CC) and up to 35% of head and neck squamous cell carcinoma (HNSCC) [2]. The incidence of HPV-induced cancers is still increasing, particularly oropharyngeal cancers [3, 4]. A significant reduction in HPV-driven carcinomas due to preventive vaccination is expected after 2050 [5].

Of the more than 200 HPV types identified, approximately 15 types are considered to be high-risk with respect to malignant tumor development [6]. The viral proteins E6 and E7 are the major viral oncoproteins, but the oncogenic potential of the E5 protein has also been recognized [7]. These viral oncoproteins interact with numerous cellular proteins and influence various cancer hallmarks [7, 8]. Integration of the HPV genome into the host DNA is considered an important step in carcinogenesis, where the viral E2 gene is usually disrupted and the expression of the E6 and E7 oncogenes is enhanced [9]. In addition, the expression of cellular genes in the vicinity of an integration site may be altered, thereby supporting carcinogenesis [10].

Ren et al. characterized an alternative HPV carcinogenesis pathway that is not dependent on E6/E7 expression and viral genome integration but is driven by episomal expression of the E2, E4, and E5 genes [11]. They identified this subtype of carcinogenesis in approximately half of HPV-positive cervical and pharyngeal cancers, demonstrated activation of fibroblast growth factor receptor signaling, and verified alternative carcinogenesis in in vitro and in vivo models. The effect of the E2/E4/E5 pathway on cell proliferation and survival was p53 dependent, which may be mediated by E2 binding [12, 13]. In HNSCC, patients with E2/E4/E5 carcinogenesis had a slightly worse prognosis than those with E6/E7 carcinogenesis, but the difference was not significant.

Since immunotherapy is increasingly used against cancer, the immunological characteristics of tumors are being studied to identify prognostic and predictive biomarkers and therapeutic targets. HPV proteins affect both innate and adaptive immune responses and contribute to tumor escape from host immunity [7, 14]. Therefore, the different levels of HPV oncoproteins associated with the two alternative pathways of carcinogenesis may provide a basis for different therapeutic targets and responses to immunotherapy. To reveal possible immunological variance between tumors with high or low E2/E4/E5 expression, we performed bioinformatics analysis of transcriptomic datasets of HNSCC and CC samples.

Identification of molecular subtypes of tumors reveals biological differences that have important implications for cancer prognosis and treatment. In HNSCC, these studies have mostly focused on differences between HPV⁺ and HPV⁻ tumors, but heterogeneity in HPV⁺ tumors has also been investigated [32]. Two basic molecular subtypes have been identified in HPV⁺ tumors of both HNSCC [33‐35] and CC [36] tumor types: (i) immune strong and (ii) highly keratinized (in terms of the reference [35]). A meta-analysis of 11 studies stratified highly keratinized HNSCC tumors into two subtypes: (i) epithelial-mesenchymal transition (EMT)-related (with high stromal score and hypoxia) and (ii) proliferation-related (with low stromal score) [37]. This classification has prognostic significance, with the best OS in patients with immune-related tumors and the worst OS in patients with EMT-related tumors. Similarly, CC patients with a strong immune response exhibited superior survival [36]. Several other bioinformatics analyses of the CC transcriptomic dataset from TCGA showed the prognostic significance of the expression of immune-related genes and/or deconvoluted levels of infiltrating immune cells [38‐43].

In our study, we aimed to investigate possible differences in the expression of immune-related genes between groups of HPV⁺ tumors with alternative carcinogenesis pathways distinguished by the level of E2/E5 expression [11]. Since the E2 and E5 proteins influence the expression and function of various human proteins involved in both innate and adaptive immunity, the type of carcinogenesis may be associated with different immune characteristics that influence patient prognosis and response to immunotherapy. To improve the homogeneity of the sample groups, we focused on HPV16⁺ CC and HNSCC tumors and excluded nonsquamous tumors from the CC dataset. Unsupervised clustering identified clusters 1 with high expression of immune-related genes and clusters 2 characterized by keratinization, consistent with clustering in previous studies [33‐36]. Comparisons between these clusters were used to evaluate differences between tumors with high or low expression of the E2/E5 genes.

The proportion of E2/E5-high tumors was comparable in cluster 1 and cluster 2 (60% and 49%, respectively) in CC, but it was predominant in cluster 1 (93%) and lower in cluster 2 (39%) in HNSCC. Higher expression of E2 and E5 genes (associated with lower integration) in a cluster of tumors with a high immune response was also found in another cohort of HNSCC [35]. Despite this difference in E2/E5 expression between CC and HNSCC, which was accompanied by a difference in immune cell infiltration, OS was comparably better in E2/E5-high tumors in both cancer types.

In the immune-related genes, we took a closer look at the genes that encode proteins that are critical for immune cell infiltration (chemokines and their ligands) and the efficacy of antitumor immunity (immune checkpoints and components of APP pathways). Comparison of HPV⁺ and HPV⁻ CCs has shown increased expression of APP genes and genes encoding immune checkpoints and markers of immune cells in HPV⁺ tumors [44, 45]. Since the E7 oncoprotein downregulates the expression of MHC class I genes and these genes were also upregulated in HPV⁺ tumors, the increased expression of the followed genes in HPV⁺ tumors was probably associated with higher immune cell infiltration and interferon (IFN)-γ production. Similarly, differences in the expression of immune-related genes found in our analysis between samples from clusters 1 and 2 and tumors with high and low E2/E5 expression corresponded to the levels of immune infiltrating cells, and we did not find any gene with significantly different expression between E2/E5 high and E2/E5 low tumors that could be attributed to the level of E2 or E5 expression (i.e., an immune-related gene without differential expression between clusters 1 and 2).

Downregulation of APP pathway components is a well-known mechanism of tumor immune escape that can contribute to tumor progression and lead to resistance to cancer immunotherapy, including blockade of PD-1/PD-L1 signaling [46]. As confirmed by our analysis, low expression of APP components, which is associated with worse prognosis, is common in CC and HNSCC. Therefore, approaches of cancer immunotherapy that take into account this downregulation should be applied against such tumors [47‐49]. In tumors with reversible MHC class I downregulation, this expression can be restored by the induction of IFN signaling [47]. However, stimulation of IFN pathways also upregulates compensatory mechanisms that prevent autoimmune damage of tissues. In our analysis, we found increased expression of immune checkpoints in tumors with high immune cell infiltration, and we also noticed high expression of 11 leukocyte immunoglobulin-like receptor (LILR) genes. This family encodes both activating (LILRA1-6) and inhibitory receptors (LILRB1-5) [50]. Some LILR members bind MHC class I molecules. For example, LILRB1 binding is dependent on the expression of β-2 microglobulin and provides a ‘don’t eat me’ signal to macrophages [51]. Since LILRB1 is also expressed on NK cells and its engagement of MHC class I molecules (including nonclassical HLA-G molecules) inhibits cell lysis by NK cells, LILRB1 blockade could be used for immunotherapy of both MHC class I-proficient and MHC class I-deficient HPV-driven tumors.

In our study, we did not find an association of E2 or E5 expression with the immune characteristics of CC and HNSCC tumors or patient survival. Rather, the expression of E6 and E7 genes associated with HPV DNA integration seems to be a major viral factor affecting tumor progression and severity. When the expression of 7 HPV16 proteins (L1, L2, E1, E2, E5, E6, E7) was analyzed in CC, only E6 and E7 were significantly associated with OS, and significantly increased E6 activity was detected in the cluster of patients with a high immune response [35, 36], which is consistent with the finding that the E6 and E7 oncoproteins inhibit keratinocyte differentiation, including keratinization [52, 53]. In addition, spliced E6* isoforms, which are associated with poorer prognosis, were increased in the high keratinization cluster [35].

Although we did not find immune-related genes differentially expressed in E2/E5-high versus E2/E5-low tumors in this analysis of transcriptomic datasets, the level of HPV proteins may affect the level and function of some cellular immune-related proteins, which we were not able to detect in our analysis. For example, the cyclic GMP–AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway, that can be critical for protection against infection and in cancer immunotherapy [54] can be inhibited by several HPV proteins. While the HPV18 E7 protein can directly bind STING [55] and inhibit downstream NF-κB signaling [56], the HPV16 E7 protein destabilizes STING via the mitochondrial NOD-like receptor family member X1 (NLRX1) binding [57]. In addition, both HPV16 and HPV18 E7 proteins downregulate STING and cGAS expression by upregulating the histone methyltransferase SUV39H1 [58]. The E5 protein has also been shown to bind STING and inhibit downstream IFN signaling [59] and the E2 protein downregulates STING and IFN-κ expression [27]. Finally, the E6 protein binds and inhibits the interferon regulatory factor-3, a component of the STING pathway [60]. Suppression of STING signaling limits the effect of STING agonists in the treatment of HPV-associated tumors [59, 61, 62], but the relative contribution of individual HPV proteins and the difference between E2/E5-low and E2/E5-high tumors have not been investigated.

Finally, the E2 and E5 oncoproteins themselves could be used as targets for vaccination. Therapeutic vaccines against HPV-associated tumors are usually based only on the E7 and/or E6 oncoproteins, which are considered indispensable for maintaining the malignant transformation of cells [63]. However, the detection of specific immunity against HPV in HNSCC has shown a broad response of CD4 and CD8 T cells against viral antigens E1, E2, E4, E5, E6, E7, and L1 [64], and E1, E2 and E5 have been identified as major targets of intratumoral CD8 T cells [65, 66]. T-cell responses to E2 are also common in cervical premalignant and malignant lesions [67, 68]. Vaccination against E2 may be particularly beneficial in patients with E2/E5-high tumors. For example, a virus-based vaccine carrying conserved elements of HPV16/18/31/52/58 E1/E2/E4/E6/E7 could be applied [69].

Our results suggest that while the regulation of expression of immune-related genes by the viral E2 and E5 proteins, which was found in transfected/transduced keratinocytes, is probably important for evasion of the immune system and reproduction of HPVs in nonmalignant lesions, expression of E2 and E5 is not the major determinant of immunological characteristics of tumors, especially in CC. The major role of E2 and E5 expression in carcinogenesis may be in deregulation of the E6/E7 oncogenes after E2 downregulation and/or modulation of the function of some cellular proteins.