Involvement of HPV Infection in the Release of Macrophage Migration Inhibitory Factor in Head and Neck Squamous Cell Carcinoma

Abstract

Human papilloma virus (HPV) infection has been well-established as a risk factor in head and neck squamous cell carcinoma (HNSCC). The carcinogenic effect of HPV is mainly due to the E6 and E7 oncoproteins, which inhibit the functions of p53 and pRB, respectively. These oncoproteins could also play a role in the Warburg effect, thus favoring tumor immune escape. Here, we demonstrated that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is expressed at higher levels in HPV-negative patients than in HPV-positive patients. However, the secretion of MIF is higher in HPV-positive human HNSCC cell lines, than in HPV-negative cell lines. In-HPV positive cells, the half inhibitory concentration (IC₅₀) of MIF inhibitor (4-iodo-6-phenylpyrimidine (4-IPP)) is higher than that in HPV-negative cells. This result was confirmed in vitro and in vivo by the use of murine SCCVII cell lines expressing either E6 or E7, or both E6 and E7. Finally, to examine the mechanism of MIF secretion, we conducted proton nuclear magnetic resonance (¹H-NMR) experiments, and observed that lactate production is increased in both the intracellular and conditioned media of HPV-positive cells. In conclusion, our data suggest that the stimulation of enzymes participating in the Warburg effect by E6 and E7 oncoproteins increases lactate production and hypoxia inducible factor 1α (HIF-1α) expression, and finally induces MIF secretion.

Keywords: 4-IPP; HNSCC; HPV; MIF; metabolism.

Figure 1

Intracellular migration inhibitory factor (MIF) expression in head and neck cancer patients. (A) Quantitative analysis of MIF expression in a series of 39 oropharyngeal cancer patients, including 21 Human Papilloma Virus negative (HPV-ve) cases and 18 HPV+ve cases (p = 0.001, Kruskal–Wallis test) and (B) 117 oral cavity cancer patients, including 65 HPV-ve cases and 52 HPV+ve cases (p = 0.004, Kruskal–Wallis test). (C,D) Immunohistochemistry of MIF in HPV-ve (C) and HPV+ve (D) oropharyngeal cancer cases and (E,F) in HPV-ve (E) and HPV+ve (F) oral cavity cancer cases.

Figure 2

Relative MIF messenger RNA (mRNA), protein expression and macrophage migration inhibitory factor (MIF) concentration in culture medium from mouse and human culture cell lines and the response to MIF inhibitor. (A) Upregulation of MIF mRNA in human papilloma virus (HPV)-positive cell lines compared to the MIF expression in HPV-negative cell lines (p <0.001, Student’s t-test). (B) Western blot analysis showing the MIF expression in HPV-ve and +ve cell lines. (C) Increased MIF level in the culture medium of HPV+ve cells (n = 3) compared to the MIF level in the culture medium of HPV-ve cells (n = 3) (p = 0.04, Student’s t-test). (D) Concentration of 4-iodo-6-phenylpyrimidine (4-IPP) required to achieve 50% inhibition of cell proliferation in human HPV-ve and HPV+ve cell lines (p = 0.018, Student’s t-test). (E) Increased MIF levels in the culture medium of SCCVII MIFKD cells (n = 4) compared to the SCCVII MIFsc cells (n = 3) (p = 0.045, Student’s t-test).

Figure 3

Macrophage migration inhibitory factor (MIF) secretion by murine cells in vitro and in vivo, and 4-IPP IC50 in murine cell lines. (A) Increase in the MIF concentration in the culture medium of SCCVII cells expressing HPV oncoproteins (p <0.001, one-way ANOVA). (B) Percentage of cell proliferation after exposure to 50 µM 4-iodo-6-phenylpyrimidine (4-IPP) in SCCVII CT, E6, E7 and E6/E7 cell lines (p <0.001, one-way ANOVA test). (C) Increase in the serum MIF levels of mice receiving SCCVII E6/E7 cells (n = 18), compared to the serum MIF levels of mice in the control group (CT) (n = 16) (p = 0.013, Mann–Whitney test).

Figure 4

Lactate production in human cell lines. (A) Partial least-squares discriminant analysis (PLS-DA) for human papilloma virus (HPV)-positive and HPV-negative cell lines. Increase in lactate production in (B) the extracellular compartment (p <0.001, Mann-Whitney test) and in (C) the intracellular compartment (p = 0.024, Mann-Whitney test) of HPV-positive and HPV-negative cells.

Figure 5

Hypoxia inducible factor 1α (HIF-1α) expression in human and murine cell lines. (A) Western blot analysis demonstrating the upregulation of HIF-1α in two human papilloma virus (HPV)-positive cell lines (UPCI-SCC-090 and 93VU147T) and in the murine SCCVII E6/E7 cell line. Concentration of macrophage migration inhibitory factor (MIF) in the culture medium of (B) human HPV-negative cell lines (p <0.001, Student’s t-test), and (C) in human positive cell lines (N.S., Student’s t-test) under normoxic and hypoxic conditions.

Figure 6

Schematic illustration of the role of human papilloma virus (HPV) oncoproteins in the production of macrophage migration inhibitory factor (MIF). The oncoprotein E6 promotes the activation of the mTOR signaling pathway, thus leading to the upregulation of hypoxia inducible factor 1α (HIF-1α), which enhances the production of MIF and increases the production of lactate. The oncoprotein E7 drives the accumulation of the dimeric form of pyruvate kinase M2 (PKM2), thus leading to the conversion of pyruvate to lactate through lactate dehydrogenase (LDHA). Moreover, PKM2 interacts with HIF-1α to amplify the expression of HIF-1α target genes, including MIF and LDHA. Additionally, the HPV E2 protein localizes to the mitochondrial membrane, leading to the increased production of reactive oxygen species (ROS), which increases the stability of HIF-1α. The enrichment of MIF in the extracellular environment could promote the tumor immune escape by the accumulation of pro-tumoral immune cells such as tumor-associated macrophages (TAM), and by the decrease in antitumoral cells such as cytotoxic T cells, Langerhans/dendritic cells (DC), and natural killer (NK) cells. Complete arrows indicate data from the literature, and the red dotted arrow indicates a hypothesis.