SARS-CoV-2 infection become a new public health crisis across the world with the emergence and rapid spread of coronavirus disease 2019 (COVID-19). Since cancer patients are known to be immunocompromised, they are considered a distinct vulnerable population with a high risk of COVID-19-associated complications (Yang et al. 2020; Desai et al. 2021). For instance, SARS-CoV-2 infection led us to reduce elective surgeries due to a lack of knowledge on the impact of COVID-19 disease on cancer patients (COVIDSurg Collaborative 2020b). However, a major problem put forth by physicians/clinicians is that a delay or cancellation of surgery may directly or indirectly result in poor outcomes and increased fatality. For instance, reports have suggested that a delay in proper care for a patient with mild or less aggressive forms of HNSC resulted in pulmonary complications associated with the SARS-CoV-2 virus (COVIDSurg Collaborative 2020a). Though studies suggested the possibility of suspending surgery in a few cases, the main therapeutic strategy for these tumors is surgery (Brody et al. 2020). In this view, the potential impact of COVID-19 on the pattern of presentation of HNSC patients needs further exploration to provide optimal care with efficient treatment. Thus, we hereby investigated the possible explanations for the increased susceptibility and mortality rate in HSNC patients infected with SARS-CoV-2.
First, we targeted the ACE2 and TMPRSS2 expression levels because they both play a vital role in viral host entry and disease pathogenesis of SARS-CoV-2. Indeed, ACE2 and TRMPRSS2 are associated with influenza, SARS-CoV, as well as SARS-CoV-2 in regulating viral entry into the host cell (Heurich et al. 2014; Simmons et al. 2013). To note, it is demonstrated that the affinity of the S1 spike protein of SARS-CoV-2 is 10–20 times higher towards ACE2 (Wrapp et al. 2020) and it is cleaved by TMPRSS2, thereby activating the endocytic route of SARS-CoV-2 (Hoffmann et al. 2020). Numerous studies have recently suggested that ACE2 and TMRPSS2 could be potential biomarkers for the early prediction of COVID-19 disease severity (Skarstein Kolberg 2020; Fagyas et al. 2022; Strope, PharmD, and Figg 2020; Rahbar Saadat et al. 2021). With this background, we evaluated the mRNA expression of ACE2 and TMPRSS2 in HNSC utilizing multidisciplinary parameters. We found that ACE2 expression was downregulated in HNSC with HPV infection, suggesting higher shedding of membrane ACE2 from head and neck region, particularly in sinuses, vocal cords, salivary gland and oral cavity, since these regions are known to express higher levels of ACE2 (Descamps et al. 2020). On the other hand, though we observed reduced expression of TMRPSS2 in HNSC, its mRNA levels were found substantially elevated in HNSC associated with HPV. In reference to this, a recent study reported that the reduction of TMPRSS2 was more evidence in HNSC patient with shorter survival as well as those with HPV negative status. (Sacconi et al. 2020). This indicates the virus or any viral factors/viral-induced host mediators could be involved in modulating TMPRSS2 expression, which leads to higher expression of TMPRSS2 and aids in priming of S protein for host viral entry as well as increased disease severity.
Since both the genes mediate sex-specific effects (Baratchian et al. 2021), we explored their expression levels in terms of gender. A differential expression of ACE2 in males and females indicates sex different expressions of ACE2 due to the effect of sex hormones, thereby contributing toward gender disparity in morbidity and mortality from COVID-19 disease. In contrast, TMPRSS2 expression was completely downregulated in both male and female HNSC cases. Thus, studies regarding the impact of sex hormones on the sex-specific expression of these genes need further validation as suggested by Majdic, 2020 (Majdic 2020). Furthermore, amplified expression of ACE2 expression in individual cancer stage-2 and age group (61–80) suggests that this patient needs effective management during SARS-CoV-2 infection because early studies have reported that people over the age of 40 are at high risk for HNSC (Schantz and Yu 2002). In alignment with this, our results showed an amplified expression of ACE2, particularly in individual cancer stage-2 and age group (61–80) compared to normal. However, the lack of TMPRSS2 expression in all cancer stages and age groups suggests that other host proteases may be involved in the proteolytic cleavage of the S protein of SARS-CoV-2 infection. In support of this, new scientific findings have reported the involvement of various other host proteases (furin, matriptase, cathepsin B, cathepsin L) in proteolytic cleavage of coronavirus spike proteins (Jaimes, Millet, and Whittaker 2020; Papa et al. 2021; Strope, PharmD, and Figg 2020). To note, furin, as well as cathepsin, are a few upregulated proteins associated with carcinoma invasion and progression in oral cancer patients (López de Cicco et al. 2002; Kawasaki, Kato, and Mizuno 2002). In reference to this, an in silico analysis by Zhong et al 2020 demonstrated that SARS-CoV-2 could invade the oral mucosal cell via the ACE2 receptor and interact with the cell membrane through the activation of furin protease (Zhong et al. 2020). From this, we could speculate that oral mucosa tissue is more susceptible to SARS-CoV-2, thereby enhancing the disease severity in HNSC patients. Though ACE2 and TMPRSS2 expression were downregulated in HNSC cases compared to normal, our correlation analysis showed a significant positive association between ACE2 and TMPRSS2 in HNSC patients, which emphasizes the importance of these molecules in the disease severity of HNSC and/or COVID-19 patients. This initial assessment provides a piece of primary evidence that differential expression of ACE2 and TMPRSS2 is one of the plausible explanations for the higher susceptibility and mortality rate of HNSC patients with COVID-19.
To provide strong evidence, we further investigated the functional assessment of ACE2 and TMPRSS2. In this, we developed a protein-protein interaction network among the topmost significant 40 proteins associated with COVID-19 as well as HNSC, which includes ACE2 and TMPRSS2. We observed 40 nodes of interaction, where the major interaction of nodes was noted with ACE2 and TMRPSS2. This indicates the functional involvement and substantial role of ACE2 and TMPRSS2 in the disease progression of HNSC patients with SARS-CoV-2 infection. Besides, we also characterized the mutation and copy number alteration in their respective protein sequences based on 12 studies. In total, we noted 5 mutations at five different locations of the ACE2 protein, where the highest frequency of alteration is 3.21%. Whereas, 4 mutations at 4 different locations were determined against the TMPRSS2 protein sequences, where the maximum level of alteration frequency was 1.7%. Importantly, shallow deletion was the most frequent type of CNA observed in both protein sequences. This result supports the significant involvement of ACE2 and TMRPSS2 in HNSC and COVID-19 disease progression.
Finally, we assessed the functional and molecular activities of various genes associated with HNSC and COVID-19. We found that 19% of genes were associated with catalytic activity, indicating TMPRSS2 and other host protease enzymes may be involved in proteolytic cleavage of S protein of SARS-CoV-2 for host viral entry as well as active shedding of membrane/cellular bound ACE2. This could be a possible explanation for direct release and elevated levels of ACE2 in circulation, which is shown to be strongly associated with COVID-19 disease severity (Fagyas et al. 2022). Besides TMPRSS2, other enzymes such as TMPRSS11D, HNP/TMPRSS1, ADAM17, and ADMA10 are also shown to cleave the ACE2 receptor (SENAPATI et al. 2021; Jia et al. 2009). In terms of cellular function and pathway, most of the genes were associated with cell growth, cell proliferation, inflammation, and various cancer signaling (P53, PI3K, RAS, EGFR, JAK/STAT, angiogenesis, interleukin mediated, Wnt signaling) pathways, indicating the higher possibility of HNSC progression. This was ascertained in early studies, which reported the potential involvement of underlying signaling pathways in HNSC (de Bakker et al. 2021; Thomas et al. 2015; Alamoud and Kukuruzinska 2018; Mock et al. 2021). Additionally, studies have described pro-inflammatory mediators as a crucial factor for HNSC development (Bonomi et al. 2014; Astradsson et al. 2019). Moreover, genes associated with inflammation and vascular functions show that elevated levels of pro-inflammatory mediators accompanied by hyper-inflammatory responses may be directly or indirectly involved in epithelial/endothelial dysfunction, which ultimately destroys the pulmonary endothelial cells as well as the oral cavity, leading to multi-organ failure and death, as observed in COVID-19 patients (Pelaia et al. 2020; Jin et al. 2020; Fodor et al. 2021). Hence, further investigations are required to ascertain the role of ACE2 and TMPRSS2 as biomarkers or therapeutic targets for HNSC patients to provide effective treatment and triage.