2. Interferons: An Overview with Attention to Cancer Surveillance
Discovered in 1957, interferon (IFN) earned its name with the recognition that cells challenged by attenuated influenza A virus created a substance that “interfered with” a subsequent infection by a live virus [14]. IFN is now understood to represent a very large family of immune-modulating proteins, divided into three types, designated as type I, II, and III based upon the receptors each IFN interacts with. Type I IFN includes both IFN-α and IFN-β, and this type is the most diverse, being further divided into seventeen subtypes. IFN-α alone has thirteen subtypes currently identified, and each of those is further divided into multiple categories [15]. Type I IFNs play a powerful role in the immune response to multiple stressors. In fact, they have enjoyed clinical therapeutic value as a treatment option for a variety of diseases and conditions, including viral infections, solid tumors, myeloproliferative disorders, hematopoietic neoplasms and autoimmune diseases such as multiple sclerosis [16].
As a group, IFNs play exceedingly complicated and pleiotropic roles that are coordinated and regulated through the activity of the family of IFN regulatory factors, or IRFs [17]. IRF9 is most directly involved in anti-viral as well as anti-tumor immunity and genetic regulation [18-20].
Closely related to this are plasmacytoid dendritic cells (pDCs), a rare type of immune cell that circulate in the blood but migrate to peripheral lymphoid organs during a viral infection. They respond to a viral infection by sharply upregulating production of type I IFNs. The IFN-α released in the lymph nodes induces B cells to differentiate into plasmablasts. Subsequently, interleukin-6 (Il-6) induces plasmablasts to evolve into antibody-secreting plasma cells [21]. Thus, IFNs play a critical role in both controlling viral proliferation and inducing antibody production. Central to both antiviral and anticancer immunity, IFN-α is produced by macrophages and lymphocytes when either is challenged with viral or bacterial infection or encounters tumor cells [22]. Its role as a potent antiviral therapy has been recognized in the treatment of hepatitis C complications [23], Cytomegalovirus infection [24], chronic active ebola virus infection [25], inflammatory bowel disease associated with herpes virus infection [26], and others.
Impaired type I IFN signaling is linked to many disease risks, most notably cancer, as type I IFN signaling suppresses proliferation of both viruses and cancer cells by arresting the cell cycle, in part through upregulation of p53, a tumor suppressor gene, and various cyclin-dependent kinase inhibitors [27,28]. IFN-α also induces major histocompatibility (MHC) class 1 antigen presentation by tumor cells, causing them to be more readily recognized by the cancer surveillance system [29,30]. The range of anticancer effects initiated by IFN-α production is astounding and occurs through both direct and indirect mechanisms. Direct effects include cell cycle arrest, induction of cell differentiation, initiation of apoptosis, activation of natural killer and CD8+ T cells, and others [31].
The indirect anticancer effects are predominantly carried out through gene transcription activation of the Janus kinase signal transducer and activator of transcription (JAK/STAT) pathway. IFN-α binding on the cell surface initiates JAK, a tyrosine kinase, to phosphorylate STAT1 and STAT2 [32]. Once phosphorylated, these STATs form a complex with IRF9, one of a family of IRFs that play a wide range of roles in oncogene regulation and other cell functions [33]. It is this complex, named IFN-stimulated gene factor 3 (ISGF3), that translocates to the cell nucleus to enhance the expression of at least 150 genes [31]. IRF9 has been suggested to be the primary member of the IRF family of proteins responsible for activation of the IFN-α antiproliferative effects, and that appears to be through its binding to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 1 and 2 (TRAIL-R1/2) [34]. IRF7 is another crucial member of the IRF family of proteins involved early in the response to a viral infection. It is normally expressed in low amounts but is strongly induced by ISGF3. IRF7 also undergoes serine phosphorylation and nuclear translocation to further activate the immune response. IRF7 has a very short half-life, so its gene-induction process is transient, perhaps to avoid overexpression of IFNs [35].
Once TRAIL is bound by IRF9, it is then able to act as a ligand for Death Receptor 4 (DR4) or DR5, initiating a cascade of events involving production of caspase 8 and caspase 3, and ultimately triggering apoptosis [36]. Dysregulation of this pathway, through suppression of either IFN-α or IRF9 and the resulting failure to bind TRAIL-R, has been associated with several hematologic malignancies [37], and has been shown to increase the metastatic potential in animal models of melanoma, colorectal cancer, and lymphoma [38].
IFN-α both initiates and orchestrates a wide range of cancer suppressing roles. Dunn et al. (2005) showed that IFN-α plays an active role in cancer immunoediting, its locus of action being hematopoietic cells that are “programmed” via IFN-α binding for tumor surveillance [39]. It is via the exceedingly complex interactions between type I IFNs and IRF7 and IRF9 in particular that a great deal of antiproliferative effects are carried out. This is evidenced by the large number of studies showing increased tumor growth and/or metastases associated with a wide number of cancer types.
For example, Bidwell et al. (2012) found that, among over 800 breast cancer patients, those with high expression of IRF7-regulated genes had significantly fewer bone metastases, and they propose assessment of these IRF7-related gene signatures as a way to predict those at greatest risk [40]. Use of microRNA to target IRF7 expression has also been shown to enhance breast cancer cell proliferation and invasion in vitro [41]. Zhao et al. (2017) found a similar role for IRF7 in relation to bone metastases in a mouse model of prostate cancer [42]. Regarding the anti-cancer mechanism behind IRF7 expression, Solis et al. (2006) found that IRF7 induces transcription of multiple genes and translation of their downstream protein products including TRAIL, IL-15, ISG-56 and CD80, with the noted therapeutic implications [43].
IRF9, too, has a central role to play in cancer surveillance and prevention. Erb et al. (2013) demonstrated that IRF9 is the mediator through which IL-6 augments the anti-proliferation effects of IFN-α against prostate cancer cells [44]. Tian et al. (2018) found IRF9 to be a key negative regulator of acute myeloid leukemia cell proliferation and evasion of apoptosis [45]. It does so, at least in part, through acetylation of the master regulatory protein p53.
Both IFN-α and IRF9 are also apparently necessary for the cancer-preventative properties of a fully functional BRCA2 gene. In a study presented as an abstract at the First AACR International Conference on Frontiers in Basic Cancer Research, Mittal and Chaudhuri (2009) describe a set of experiments which show for the first time that BRCA2 expression leads to increased IFN-α production and augments the signal transduction pathway resulting in the complexing of IRF9, STAT1 and STAT2 described previously [46]. Two years prior, Buckley et al. (2007) had established that BRCA1 in combination with IFN-γ promotes type I IFNs and subsequent production of IRF7, STAT1, and STAT2 [47]. Thus, the exceedingly important cancer regulatory genes BRCA1 and BRCA2 rely on IRF7 and IRF9, respectively, to carry out their protective effects.
In a preprint, Mamoor (2020) used gene expression analysis to determine that infection with either SARS-CoV-1 (in mice) or MERS-CoV (in vitro ) leads to increased production of IRF7 and IRF9, and the author speculates that “IRF7 and IRF9 may be important for SARS-CoV-2 immune defense in humans.” [48] This speculation is somewhat confirmed by Rasmussen et al. (2021), who reviewed the compelling evidence that deficiencies of either IRF7 or IRF9 lead to significantly greater risk of severe COVID-19 illness [49]. Importantly, they also note that evidence suggests type I IFNs play a singularly important role in protective immunity against COVID-19 illness, a role that is shared by multiple cytokines in most other viral illnesses including influenza.
As will be discussed in more detail below, the SARS-CoV-2 spike protein modifies host cell exosome production. Transfection of cells with the spike gene and subsequent spike protein production results in those cells generating exosomes containing microRNAs that suppress IRF9 production while activating a range of pro-inflammatory gene transcripts [50]. Since these vaccines are specifically designed to induce high and ongoing production of spike proteins, the implications are ominous. As described above, inhibition of IRF9 will suppress TRAIL and all its regulatory and downstream apoptosis-inducing effects. IRF9 suppression via exosomal microRNA should also be expected to impair the cancer-protective effects of BRCA2 gene activity, which depends on that molecule for its activity as described above. BRCA2-associated cancers include breast, fallopian tube, and ovarian cancer for women, prostate and breast cancer for men, acute myeloid leukemia in children, and others [51].
Vaccination has also been demonstrated to suppress both IRF7 and STAT2 [52]. This can be expected to interfere with the cancer-protective effects of BRCA1 as described above. Cancers associated with impaired BRCA1 activity include breast, uterine, and ovarian cancer in women; prostate and breast cancer in men; and a modest increase in pancreatic cancer for both men and women [53].
Reduced BRCA1 expression is linked to both cancer and neurodegeneration. BRCA1 is a well-known breast cancer susceptibility gene. BRCA1 inhibits breast cancer cell proliferation through activation of SIRT1 and subsequent suppression of the androgen receptor [54]. In a study conducted by Suberbielle et al. (2015), reduced levels of BRCA1 were found in the brains of Alzheimer’s patients [55]. Furthermore, experiments with knocking down neuronal BRCA1 in the dentate gyrus of mice showed that DNA double-strand breaks were increased, along with neuronal shrinkage and impairments in synaptic plasticity, learning and memory.
Analysis detailed in a recent case study on a patient diagnosed with a rare form of lymphoma called angioimmunoblastic T cell lymphoma provided strong evidence for unexpected rapid progression of lymphomatous lesions after administration of the BNT162b2 mRNA booster shot [56]. Comparisons of detailed metrics for hypermetabolic lesions conducted immediately before and 21 days after the vaccine booster revealed a five-fold increase after the vaccine, with the post-booster test revealing a 2-fold higher activity level in the right armpit compared to the left one. The vaccine had been injected on the right side. It is worth pointing out in this regard that lymphoid malignancies have been associated with suppression of TRAIL R1 [57].
Given the universally recognized importance of optimally functioning BRCA1/2 for cancer prevention and given the central role of the TRAIL signal transduction pathway for additional cancer surveillance, the suppression of IRF7 and IRF9 through vaccination and subsequent spike protein production is extremely concerning for long-term cancer control in injected populations.