Benefits and Pitfalls of a Glycosylation Inhibitor Tunicamycin in Therapeutic Implication of Cancers

1. Introduction

Protein glycosylation is a unique physiological and pathophysiological process that attaches glycans (carbohydrate-based polymers) to the protein backbone via N-linked glycosylation (at the asparagines or glutamines carboxamide side chain) or O-linked glycosylation (at the hydroxyl group of serine/threonine side chains) [1,2]. Glycosylation is the most frequent post-translational modification of a protein [3] and the key molecular event associated with innate and adaptive immune systems [4]. It is involved in cell membrane formation, folding and unfolding, and protein stability [5]. Thus, the functional utilities of protein glycosylation in health and diseases are growing interest in biomedical research [4,6].

The aberrant glycosylation with unique structures, known as tumor-associated carbohydrate antigens (TACAs) [7,8,9], plays a vital role in malignant transformation, cancer progression, and metastasis [10,11]. Aberrant Glycosylation, particularly that associated with enzymes, is useful as a biomarker for cancer cells. The enzymes that cause aberrant glycosylation express cancer-specific changes, especially over-expression of certain enzymes in cellular biosynthesis. For example, in the proteomics study, analysis of the glycans and glycoproteins in cancer and normal cells can yield personalized diagnosis and treatments, partly because cancer cells' metabolism differs from normal cells. By continuing to study the cancer-modified glycans, these biomarkers can better be used to detect and diagnose specific types of cancer [12]. The spreading of primary cancer cells to distant organs is coupled with alterations and degradation of the extracellular matrix (ECM), impacting cellular interactions and structural changes of surface proteins by glycosylation [4,5,6,13,14,15,16]. Thus, glycosylation is now reconsidered in the era of cancer-targeted therapy [17]. Thereby, particular emphasis on glycosylation inhibitors is given for the rational development of immunologically targeted cancer therapies.

Tunicamycin (TM), a mixture of homologous nucleoside antibiotic inhibitors of N-linked glycosylation, may inhibit drug resistance in multidrug therapy. This drug impedes the transfer of UDP-N-acetylglucosamine (GlcNAc) to dolichol phosphate in the endoplasmic reticulum (ER) of eukaryotic cells, thus disrupting protein maturation by blocking oligosaccharides biosynthesis [18]. Oligosaccharides are instrumental in N-linked glycosylation, linking to nitrogen atoms from asparagine residue. The formulation and structure of proteins depend on this natural process. The inhibition of this process results in unfolded protein response and cellular stress, therefore resulting in cell death. However, as effective as it is, this stalwart offensive of canceling N-linked glycosylation is not cell-type specific. TM in cancer treatment could prove hazardous, as residual cytotoxicity on surrounding normal tissue could worsen the state of the disease. Nanocarriers, a prospective asset in the pharmaceutical world, may present a solution to hinder this drug's cytotoxicity.

2. Origin and Basic Biology of Tunicamycin

Tunicamycin (TM) is a nucleoside antibiotic consisting of tunicamine, uracil, N-acetylglucosamine, and fatty acyl side chain (Figure 1). Streptomyces lysosuperificus, classified by Tamura et al. in 1970, is the sole producer of this antibiotic [19]. This bacterium was cultivated in a fluid of 2% glucose, 2% gluten meal, 2% pharma media, 3% K₂HPO_4, 1.5% KH₂PO₄, and 0.3% CaCO₃. After 30 hours in the culture broth, TM was detected as a product of fermentation of streptomyces lysosuperificus and accumulated over 100 hours. Newcastle disease virus was used as a subject to test the antibiotic activity. TM exhibits antibacterial activity due to its inhibition of phospho-MurNAc-pentapeptide translocase (MraY), an enzyme essential in bacterial cell wall synthesis [20]. MraY is classified as a translocase that helps in cell wall biosynthesis utilizing UDP-MurNAc, a pentapeptide necessary in peptidoglycan synthesis. TM inhibits MraY similarly to how it blocks dolichol phosphate in N-linked glycosylation. In a study conducted by Duskin et al., three variants of 3T3 fibroblasts were treated with TM: normal 3T3 cells, SV40-transformed 3T3 cells, and polyomavirus-transformed 3T3 cells [21]. TM was administered to variants in cell plates and displayed high cytotoxic effects on all 3T3 cell types, differing in some factors. After 2-4 days, the cytotoxicity of TM altered the morphology of their regular epithelioid shapes into long, pointed structures. These changes were present when TM was present in all cell plates. However, when TM was removed, any physiological changes soon regressed into a normal state, proving that TM lingers around any targeted subject and applies cytotoxicity until removed from a subject.

3. How does Tunicamycin work?

A multifaceted mode of action of TM has been reported as a therapeutic drug. In any cancer treatment, the development of drug resistance seems to be almost imminent, and this limiting factor prevents achieving cures in patients with cancers [22]. The aberrant glycosylation is one of the leading factors in developing drug resistance phenotypes in cancer cells [23]. Some oncoproteins and oncogenes promote aggressive phenotypes such as proliferation and drug resistance via N-linked glycosylation [24,25]. TM inhibits N-linked glycosylation and other N-glycans via inducing ER stress and hindering the development of invasive behavior of cancer cells. These include cancer stemness and drug resistance [26]. However, it is uncertain if TM could regulate the mutant effect of proto-oncogenes associated with the above pathological events. Mechanistically, TM can enter eukaryotes via the protein transporter major facilitator domain containing 2A (MFSD2A) and permeates through the endoplasmic reticulum (ER) membrane, causing stress [27,28]. TM induces ER stress through the unfolded protein response (UPR), promoting TRAIL-induced apoptosis [29,30]. The whole genome microarray analysis found that under sustained ER stress by TM, endothelial nitric oxide synthase (eNOS) upregulated in prostate cancer cells that activated mTORC1 through eNOS-RagC pathway and promote p62-reactive oxygen species (ROS) dependent mitochondrial apoptosis [30]. The UPR work through three receptors are located on the ER membrane. These include Protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme (IRE1), and activating transcription factor 6 (ATF6) [31,32,33,34,35]. These receptors are usually found inactive in scenarios where UPR is nonexistent due to its regulation by glucose-reacting protein 78 (GRP78). The induction of TM inhibits N-acetyl glucosamine-phosphate transmission to UDP-dolichol phosphate, resulting in unfolded proteins. GRP78 then activates the three UPR mediating receptors. The cytosolic portion of ATF6 then enters the nucleus and encodes a homologous C/EBP protein (CHOP) [36]. Overexpression of this CHOP protein leads to autophagy, which inevitably leads to apoptosis (Figure 2).

4. Tunicamycin in Cancer Therapy

Despite having some issues, TM's anticancer properties, which are mediated through UPR induction due to N-linked glycosylation in eukaryotes, are well established [37,38,39]. The following sections briefly and categorically highlight TM's impact on various cancers. The in vitro and animal studies found that the most targeted tumors for Tunicamycin are the breast, liver, colon, lungs, and pancreas, depicted in Figure 3.

Breast cancer- Breast cancers (BC) with metastasis are a global threat to female health as treatment options are still limited and weakly effective. Systemic drug therapy is still the first-line option for metastatic breast cancer treatment with surgery and radiation therapy. These include hormone therapy, chemotherapy, targeted therapy, and immunotherapy. However, the current systemic drug therapies can most likely not remove all cancer cells. Thus, new drugs and therapeutic options are urgently needed. With several preclinical drugs, TM could be a potential drug for treating BC with metastasis, as preclinical studies showed TM blocks BC growth and metastasis through the regulation of the Akt/NF-kB-signaling pathway [40] and enhance trastuzumab antitumor activity on BC [41], suggesting a promising approach of combination therapy of TM and trastuzumab to improve trastuzumab's clinical efficiency [41]. The role of Wnt/β-captain in TM-induced ER-stressed mediated apoptosis in triple-negative breast cancer (TNBC) cells has been documented, providing additional TM mechanisms [42].

Cancer stem cells (CSCs) play a significant role in tumor growth and drug resistance [43]. They are characterized to have the cell surface phenotypes CD44+/CD24- [43]. About 1-4% of BC cells carry CD44+/CD24-, which is considered highly aggressive [43]. TM-induced ER stress reduces the growth and invasion of CD44+/CD24- cells, indicating TM therapy is an exciting approach to target breast cancer stem cells [44].

Colon Cancer- Colon cancer, which is also called colorectal cancer (CRC), is one of the most common digestive tract tumors and the leading cause of cancer-related death in the elderly population in the United States and globally [45]. However, early screening can find non-cancerous abnormal growths in the colon, called polyps, which can be removed (polypectomy) and prevent cancerous growth in the colon. In the advanced stage, chemotherapy is the only option before or after radiation. Targeted and immunotherapy is also considered for advanced colon cancer.

The studies suggest TN could be an anti-CRC drug as TN treatment efficiently inhibits CRC cell growth and aggressive behavior by downregulating vimentin, FIB, and Ecacollagen type I and Slug expression levels and tumor-bearing mice's survival [46]. Further, TN has been found to enhance TRAIL-induced apoptosis in CRC cells by JNK-CHOP-mediated DR5 upregulation and inhibiting the EGFR pathway [47]. TN reprogramed cell-cell adhesion in undifferentiated CRC cells by inducing E-cadherin [48]. Interestingly, separate indirect studies claimed TM is a promoting factor of CRC [49,50].

Lung Cancer- Lung cancer is the most common cancer-related death in the human population and is highly correlated with cigarette smoking [51]. The non-small cell lung cancer (NSCLC) is the primary subtype of lung cancer and accounts for about eighty percent of the new cases of lung cancer [51,52]. The therapeutic options and efficacies of NSCLC are limited due to drug resistance. Thus, the 5-year survival rate of NSCLC remains at 15% [51,52]. In lung cancer, the therapeutic roles of TN are well documented. The studies showed that TM enhances the anticancer effects of Cisplatin on lung cancer growth via deglycosylation of PTX3 through the AKT/NF-κB signaling pathway [52]. Furthermore, TM promotes anti-immunity in a lung cancer mouse model with combination therapy [53].

Pancreatic ductal adenocarcinoma (PDAC)- Among pancreatic cancers, PDAC is the deadliest cancer in the pancreas and constitutes 90% of pancreatic cancer. PDAC represents the 7^th most common cause of cancer-related death globally and has an overall 5-year survival rate not exceeding 9% [54,55]. It is anticipated that PDAC could be the second most common cause of cancer-related death in the USA by 2040 [54,56]. Based on genomic alterations, PDAC can be classified into four groups. These include stable genomes (< 50 percent structural variants per genome), scattered genomes (50-200 structural variants per genome), locally rear-ranged genomes (>200 structural variants clustered on less than three chromosomes), and unstable genomes (>200 structural variants distributed in the genome) [57,58]. This genome classification provides precision oncology knowledge and has clinical potential for PDAC patients [57].

Despite having a limited effect or no outcome of the therapy due to quick development resistance to drugs, the first-line treatments of PDAC are still FOLFIRINOX (combination of 5FU, Leucovirin, Oxaliplatin, and Irinotecan), Gemcitabine alone or in combination with nab-Paclitaxel [54,59]. The therapeutic limitations of PDAC show the urgency of finding new therapeutic options [54,59]. Mutant K-RAS in the ductal epithelial cells is the driving force of the initiation and progression of PDAC [57,60,61,62,63,64], which can be promoted by multiple factors, including CCN-family proteins [65,66]. Several preclinical studies are actively involved in discovering small molecules to target K-RAS mutants [67].

Mutant K-RAS plays a critical role in the immunosuppression of PDAC microenvironment [68,69,70]. Thus, immunologically, mutant K-RAS has an equally important target [71,72,73]. Despite having mixed and descriptive information on T-helper 2 (Th2) cells and associated cytokines in cancer, recent studies found that Th2 cells may be crucial regulators of colon and pancreas cancer progression by affecting immune cell composition and type II immune responses. In murine allograft models of colon and pancreatic cancers, the adoptive transfer of Th2 cells into tumor-bearing mice significantly reduces tumor growth by inducing cytotoxicity via eosinophils and macrophages' innate immune response. The studies also showed that IL-5 protects against tumor growth by recruiting and activating eosinophils [74]. Although glycosylation plays a vital role in regulating the above proteins, the impact of TM is underdetermined.

Overexpression of eukaryotic translational initiation factors in various cancers, including in PDAC, is a common alteration. The studies showed that one of the factors, eIF4E, is overexpressed in poorly differentiated and metastatic PDAC [54,75]. eIF4E phosphorylation (p-eIF4E) at serine209 by MNK1/2 serine/threonine kinases promotes its transformation activity [76,77]. Thus, upon mutant K-RAS activation in PDAC, eIF4E activated and promoted tumor invasion and metastasis. Studies have shown that TM treatment can inactivate eIF4E [78]. Based on these studies, we can deduce that TM treatment can be an ideal therapeutic approach for PDAC.

5. Tunicamycin in Immunotherapy

Tumor initiation and progression are linked with altered or deluded immunity [79] or reprogramming of immune cells within the tumor microenvironment [80,81]. The communication between tumor cells and the immune system is dynamic, reminiscent of a balance of immunity factors [79,80]. Thus, immunooncology is now at the forefront of basic, preclinical, and clinical research and has recently been harnessed as one of the pillars of cancer therapy [79,82,83,84,85,86]. The central principle of cancer immunotherapy is eliminating cancer cells by host cytotoxic immune CD8⁺ T cells [79,84,85,86,87]. However, this does not always happen in the tumor ecosystem, as tumor cells manipulate the system, cause tumor-specific CD8+ T cell dysfunction via antigen-derive differentiation program [88], or activate various immune checkpoint suppressive mechanisms. These include regulatory T (Treg) cells, over-expression of program death-1 (PD-1) receptor and its ligand PD-L1, CTLA-4, and others [83,84,89,90], which are briefly depicted in Figure 4.

In many different malignancies, immune-targeted therapy primarily focuses on the T-cell base [79], as the T-cells have the potency to suppress tumor growth clinically [79,87,91,92]. Despite the clinical successes of immune checkpoint inhibitors, the mechanisms underlying inhibitors' upregulation in cancer cells and infiltrating T cells have only begun to be understood. The emerging findings showed that Indoleamine 2,3-dioxygenase 1 (IDO1) is overproduced in highly tumorigenic self-renewing repopulating cancer cells [93]. IDO1 catalyzes the committing and rate-limiting step of the kynurenine (KYN) metabolic pathway, leading to abundant KYN release [93,94]. KYN is then absorbed in CD8⁺ T cells, activating the aryl hydrocarbon receptor (AhR) that causes PD-1 overexpression [93]. This sequential event adopts a tumor immunological microenvironment that is defective in recognizing and killing cancer cells [94].

The PD-L1 regulation in cancer cells is mediated by multiple pathways depending on the cancer type. These pathways are genomic alterations, epigenetic modifications, transcriptional and post-transcriptional regulation, post-translation modifications, and exosomal transport [95,96,97].

In the tumor microenvironment, the PD-1 receptor is highly N-glycosylated in T cells to maintain PD-1 stability and interaction with the PD-L1 ligand expressed in tumor cells to dampen the activity of T-cell receptor (TCR)/CD28) signals [98]. The studies showed several N-glycosylated inhibitors, including TM treatment deglycosylated PD-1 [98,99] (Figure 4). Thus, TM could be considered an ideal therapy to target PD-1 alone or in combination of other PD-1 inhibitors.

Like PD-1, PD-L1 is also highly N-glycosylated to maintain its stability and binding affinity with PD-1 in various cancers and eradicate Triple-negative Breast cancer (TNBC) by targeting glycosylated PD-L1 [100]. Thus, studies suggest targeting PD-L1 glycosylation could be an ideal therapeutic option by rationale combination of cancer immunotherapies [101,102,103,104].

Interleukins (ILs) are secreted cytokines that play vital roles in the biology of cancers and immune functions [105]. Although some ILs (i.e., IL-6, IL-10, and IL-17) boost tumor growth, some ILs may promote the immune system's antitumor response. These include IL-2, IL-12 and IL-5 [105,106]. Previous studies have shown that glycosylation plays a role in IL-12 biogenesis, but the loss of glycosylation has little or no effect on IL-12 and IL-23 secretion, heterodimerization, and biological activities [107]. IL-5 is produced by both hematopoietic and non-hematopoietic cells [108]. T-cell-derived IL-5 controls eosinophil production and their activation via JAK-STAT and RAS/Raf-ERK signaling pathways [108]. Recent studies have shown that IL-5-producing CD4-positive T cells and eosinophils collectively enhance response to immune checkpoint blockade (ICB) in breast cancer. Further, it has also been reported that ICB increased IL-5 production by CD4 T-cells elevated eosinophil production from bone marrow and infiltration that eventually enhanced ICB efficacy [106,109]. T-cell-derived IL-5 exhibits heterogeneous glycosylation with four N-glycosylation sites in the extracellular regions, and removal of N- or O-linked glycosylation on IL-5 enhances the potency of the cytokine [110], without having effect on lymphocyte proliferation in a lymphocyte proliferation assay following the treatment of glycosylation inhibitor tunicamycin (TM) [108,111]. Previously, however, it has been shown that in humans, IL-5 acts only on eosinophils and basophils for their growth, maturation and activation as these cells express IL-5 receptors [112,113,114]. Thus, the results of these studies suggest that inhibition of glycosylation in IL-5 by TM could be effective only on eosinophil production, maturation and their activation. However, further studies are warranted.

In pancreatic cancer progression, IL-5/IL-5Rα roles are possibly a double-edged sword, with both pro- and antitumor activities being discovered recently [74,115,116,117]. However, the mechanistic roles of IL-5 are still unclear. Studies have shown that TM can inhibit endoplasmic-reticulum (ER)-stress-mediated autophagy and promote the effectiveness of chemotherapies on pancreatic cancer cells [118]. Further, studies showed ER-stressed inhibitors reduce IL-5 production in the Th-2 cells (naïve CD4+ T cells) [119], which inhibit colon and pancreatic cancer growth by promoting antitumorigenic responses from macrophages and eosinophils [74]. However, the effect of ER-stressed inhibitors, including TM, on IL-5 in pancreatic cancer cells and localized eosinophils is still uncertain.

In conclusion, TM shows promise in drug-induced immunotherapy when combined with other PD-1 and PD-L1 inhibitors, such as monoclonal antibodies or various chemotherapeutics (Figure 4).

6. The Pitfalls of the Use of Tunicamycin

As much as TM promises to be an efficient drug in chemo-and immunotherapy, evidence of its cytotoxicity proves that it affects all cells, whether healthy or cancerous. Thus, TM could prove consequential if TM's residual cytotoxicity affects surrounding tissues around a tumor, possibly weakening body systems depending on where TM is inducted. Direct administration of TM in murine models of leukemia resulted in potent liver toxicity. Unfortunately, as much as TM is adequate in meeting the thresholds of chemotherapy, TM's cytotoxicity is not cell-type specific. This makes TM a liability in its free form. However, nanoparticle encapsulation technology can bypass this problem. Nanoscale particles, particularly those engineered with cell-specific ligands, can recognize specific cell types. As such, encapsulating TM inside a nanoparticle can rescue healthy cells from non-specific effects of TM. Further, chemical conjugation of TM to an antibody or to a macromolecule like PEG, can result in the formation of antibody-drug conjugates (ADCs) or polymer-drug conjugates (PDCs) respectively. Both variants can be used to augment cellular accumulation of TM inside cancer cells. Collectively, nontechnology can also provide exciting prospects that may improve TM's implications with cytotoxicity and efficiency [120,121,122,123].

7. Outlook

Tunicamycin shows promise if it is to be proven applicable in targeted therapy; however, in vivo, experiments and assays may prove the need for more research and trials in mice models before it can be approved. For such a cytotoxic drug with an effect as such, many precautions need to be taken when it comes to nanoparticle delivery. Glycotherapy modeled after a TM-focused approach can suppress the invasion of cancer cells that spread beyond the tumor mass. The UPR holds many risks as TM could have a toxic buildup in surrounding healthy tissue. It should be recalled that TM's cytotoxicity is not cell-type specific. However, the morphological effects of TM can be reversed by taking TM out of the equation, as shown earlier. The antitumorigenic effects of TM can be used in next-generation cancer therapy. Many things can go wrong as the carrier does not reach the predetermined deposition site. This method should be perfected as, with many nanocarriers, toxic buildup in the organs usually leads to more complications and may result in a weaker prognosis.