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SARS-CoV-2 S1 and RBD activate the epidermal growth factor receptor (EGFR) and its downstream signaling pathways, as well as the anti-apoptotic protein survivin

Jan 29, 2024 | About the Virus

The researchers from the United Arab Emirates and Qatar conducted this study to determine the effect of the S1 subunit of the SARS-CoV-2 spike (S) protein and its receptor-binding domain (RBD) on the activation of the epidermal growth factor receptor (EGFR) and its associated downstream signaling pathways. They hypothesized that the virus may use the EGFR expressed on epithelial lung cells as a receptor/co-receptor for its entry. The results suggest that the S1 subunit and RBD activate the EGFR and its downstream signaling pathways and increase the expression and activation of the anti-apoptotic protein survivin, which is considered a crucial marker for the activation of the survival pathway in cancer cells.

The S protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. It is composed of S1 and S2 subunits, which are separated by host cell proteases. The S1 is composed of the N-terminal domain (NTD), RBD with a receptor binding motif (RBM), and two C-terminal domains. The RBD in the S1 subunit is responsible for attachment to host cells. The SARS-CoV-2 utilizes it’s S protein, to infect the target cells in the lungs through binding with their transmembrane receptor, the angiotensin-converting enzyme 2 (ACE2). Although a large body of evidence from in vitro and in silico studies strongly supports the thesis that ACE2 is necessary for SARS-CoV-2 entry, numerous studies have revealed that other factors and/or alternative cell surface receptors may also be involved in viral entry.  https://discovermednews.com/tmem106b-protein-is-an-independent-receptor-for-sars-cov-2-entry-into-ace2-negative-host-cells/

The EGFR, which is expressed on lung epithelial cells, has been suggested as a receptor/co-receptor for viral entry. The EGFR was found to be important during influenza or Salmonella infection. It has been reported that Salmonella Rck membrane protein binds and activates EGFR and its signaling pathways, resulting in receptor/bacteria co-internalization and cell infection. The binding of EGF (epidermal growth factor) to its receptor EGFR activates several signaling intermediates, including protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), thereby leading to control of cell survival and metabolism.

Protein survivin is the smallest member of the inhibitor of apoptosis protein (IAP) family. It plays a crucial role in regulating cell division and inhibiting apoptosis by inhibiting caspase activation, and is considered a crucial marker for activating of the survival pathway in cancer cells. Expression of survivin is controlled by precise mechanisms at the transcriptional and post-translational levels. Survivin is a highly expressed in the majority of human cancers, including lung, pancreatic and breast cancers. Aberrant expression of survivin is associated with tumor cell proliferation, progression, angiogenesis, therapeutic resistance, and a poor prognosis. It has been demonstrated that survivin expression was associated with the aberrant activation of a number of receptor tyrosine kinases, including the EGFR, epidermal growth factor receptor 2 (erbB2), insulin-like growth factor-1 (IGF-1), and various cell survival signaling pathways. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747886/

About the study

The authors used cancer cell lines that are known for their expression of EGFR and differential expression of ACE-2, including ACE-2 expressing lung (A549) and colon (HT-29) carcinoma cells, and non-ACE2-expressing (HeLa) cervix adenocarcinoma cells. Cells were treated with either the full S1 protein or the RBD for 5, 15, 30, and 60 minutes, using the EGF stimulation as a positive control.

Results

Administration of S1 to ACE2-expressing lung cancer cells (A549) for a duration of 5 minutes resulted in a strong phosphorylation of EGFR, AKT, and ERK1/2, which was either comparable (EGFR and ERK1/2) or even greater (AKT) than when stimulated with the maximal dose of EGF. Under similar conditions, RBD showed little effect on EGFR phosphorylation, but significantly induced AKT and ERK1/2 phosphorylation. These findings show that EGFR was specifically targeted by the full S1 protein, but not by its RBD. Furthermore, the RBD-mediated activation of AKT and ERK1/2 was independent of EGFR activation, indicating that other molecular targets and/or intracellular mechanisms may be involved.

The authors then conducted a time-course analysis of the EGFR, AKT and ERK1/2 phosphorylation. S1 induced a rapid, but transient phosphorylation of EGFR and ERK1/2, with maximal response after 5 minutes of stimulation, whereas AKT activation was more sustained in time. The RBD did not induce clear EGFR phosphorylation, but promoted rapid and robust activation of both ERK1/2 and AKT, which remained persistent after 60 minutes of stimulation. These findings confirmed the specific activation of EGFR by S1, but not by RBD. Furthermore, S1 and RBD exhibited distinct mechanisms of AKT and ERK1/2 activation.

The researchers then employed highly ACE2-expressing HT-29 colon carcinoma cells and non-ACE2-expressing HeLa cervix adenocarcinoma cells to examine the connection between the ACE2 expression and the targeting of EGFR and its downstream signaling pathways. In the non-ACE2-expressing HeLa cells, RBD failed to induce phosphorylation of AKT or ERK1/2, irrespective of the duration of stimulation. By contrast, EGF induced phosphorylation of AKT and ERK1/2 in this cell line.

In the HT-29 colon carcinoma cells, which express ACE2 at high levels, RBD induced rapid and sustained phosphorylation of AKT and ERK1/2, similar to the results observed in the ACE2-expressing A549 cells. These results strongly support the involvement of ACE2 in the phosphorylation of AKT and ERK1/2 mediated by RBD.

The authors then examined the connection between the phosphorylation of EGFR and AKT and the induction and activation of the anti-apoptotic protein, survivin. Previous data have demonstrated that survivin expression was linked to the aberrant activation of a number of receptor tyrosine kinases, including the EGFR. The results showed that both S1 and RBD induced a time-dependent increase in survivin expression, with maximal response after 30 and 60 minutes of stimulation. The EGFR antagonist, AG1478, significantly diminished the activation of survivin mediated by S1 or RBD, demonstrating a role of EGFR.

In conclusion, these in vitro results revealed, for the first time, that SARS-CoV-2 S1 subunit and RBD activate the EGFR and its downstream signaling pathways AKT and ERK1/2, and also increase the expression and activation of the anti-apoptotic protein survivin in the lung cancer cell line. The activation of EGFR mediated by S1 was ACE2-dependent. The authors emphasized that further research is required to determine whether S protein directly binds to EGFR or not, as well as the implication of the canonical ACE2 pathway.

S1 and RBD also elicited the activation of the survival pathway, inducing the expression and activation of the anti-apoptotic protein, survivin. The authors believe that the expression and activation of the anti-apoptotic protein survivin, which is considered a crucial marker for the activation of the survival pathway in cancer cells, may be a molecular basis for the complications and severity of COVID-19 observed in cancer patients, as reported by several research groups.

This article was published in Vaccines.

Journal Reference

Palakkott, A.R. et al. The SARS-CoV-2 Spike Protein Activates the Epidermal Growth Factor Receptor-Mediated Signaling. Vaccines 2023, 11, 768. (Open Access)   https://doi.org/10.3390/vaccines11040768

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