Selinexor targets expression of metabolic genes in Merkel cell carcinoma cells

Background: Merkel cell carcinoma (MCC) is a deadly skin cancer that primarily affects the elderly and immunocompromised, with mortality rates ranging from 50% to 80%. Merkel cell polyomavirus (MCPyV) is associated with 80% of cases of MCC. The primary treatment for MCC is immune checkpoint inhibitors; however, many patients are unresponsive to or do not meet criteria for treatment. The Warburg effect has linked cancer cell survival to increased glycolytic metabolism to maintain increased cellular energy demands. While initial hypotheses suggested that increased glycolysis itself was directly upregulated and important in cancer cell proliferation, more recent ideas suggest a “moonlighting” role for glycolysis genes. In general, these “moonlighting” proteins’ non-metabolic functions are equally as important if not more important than their catalytic functions. Previous research on MCPyV-positive MCC demonstrated that selinexor targeted and decreased the expression of viral T antigens, inhibited the DNA damage response, and downregulated lipogenesis proteins. More recently, these metabolic genes have been found to regulate many oncogenes and tumor suppressors. Selinexor, an approved treatment for multiple myeloma, acts as a selective inhibitor of nuclear export by blocking exportin 1 and blocking translation of key proto-oncogenes. Objectives: Here, we report the effects of selinexor on expression of glycolytic and metabolic genes, speci�cally discussing the catalytic effects on metabolic function and their indirect non-catalytic effects. Methods: Immunoblotting quanti�ed through densitometric analysis determined the protein expression in MS-1 cell lines. T-tests were used to determine statistical signi�cance. Results: Analysis revealed highly statistically signi�cant (p<0.001) or statistically signi�cant (p<0.01) downregulations of protein expression of GLUD1, GLUT3, Hexokinase 1, PFKFB2, amphiregulin, LDHA, PDHK1, and MCT1. Conclusion: In the MCC cell line MS-1, selinexor signi�cantly downregulated expression of many genes in cellular energy metabolism and cellular proliferation in a statistically signi�cant relevant manner. These results suggest that selinexor may be a novel viable option for the treatment of MCC, but further studies in vivo and clinical trials are required to validate these �ndings


Introduction
Merkel cell carcinoma (MCC) is a rare yet lethal neuroendocrine, cutaneous cancer. 1 MCC is very aggressive with a mortality rate ranging from 50% to almost 80% that primarily affects the elderly and immunocompromised. 1 MCC carcinogenesis is primarily initiated through integration of the Merkel cell polyomavirus (MCPyV) and ultraviolet (UV)-mediated DNA damage caused by exposure to UV from sunlight. 1,2rimary treatment for loco-regional MCC includes surgical removal of the primary lesion and sentinel lymph node biopsy to determine the further course of action. 1,3In metastatic MCC, systemic treatments like chemotherapy were widely employed until 2016 when immunotherapy was shown to be a viable treatment option. 1 Immunotherapy with PD1 and PDL1 immune checkpoint inhibitors (ICIs) demonstrated better long-term e cacy over chemotherapy. 4Avelumab, an ICI, was approved by the FDA in March 2017. 4Nonetheless, a signi cant number of those with advanced-stage MCC are unresponsive to MCC or are unable to be treated by immune checkpoint inhibitors. 5linexor was FDA-approved for treatment of multiple myeloma in 2020 in combination with bortezomib and dexamethasone for patients who had received at least one prior therapy. 6This selective inhibitor of nuclear export (SINE) blocks exportin 1 (XPO1) and in turn halts translation of key proto-oncogenes which complex with the cargo protein eukaryotic initiation factor 4E (eIF4E). 7In addition, selinexor is being studied and has been studied in clinical trials in many hematologic and non-hematologic malignancies, including leukemia, gastric, bladder, prostate, breast, ovarian, skin, lung, and brain cancers. 4ncer cells, such as MCC cells, upregulate and increase the metabolism of glucose to allow for increased growth, survival, and proliferation. 8,9This increased glucose metabolism includes both an increase in glucose uptake as well as an increase in the fermentation of glucose into lactate. 8This phenomenon is known as the Warburg effect. 8nce the Warburg effect established that anaerobic glycolysis is key to cancer growth and proliferation, 10 many researchers have sought to elucidate the mechanism by which glycolysis has been linked to cancer. 10,11Glucose transporters proteins, hexokinases, pyruvate dehydrogenase kinases, and lactate dehydrogenases, are all classically upregulated by the Warburg effect.Later evidence suggests that many oncogenes and tumor suppressors like PTEN, PI3K/Akt/mTOR, HIF-1α, AMPK, p53, EGFR, ERK1/2, PMK2, RAS, and Myc all regulate and alter energy metabolism in cancer. 10,11While initial hypotheses believed that increased glycolysis itself was directly upregulated and important in cancer cell proliferation, more recent ideas suggest a "moonlighting" role for glycolysis genes. 10,11Generally, "moonlighting proteins" are proteins that exhibit more than biochemically or physiologically important function. 12"Moonlighting" within glycolysis speci cally posit that the non-metabolic functions of glycolytic enzymes are equally as important if not more important than the enzymes catalytic function in cancer progression, and the term "moonlighting" within science has been used since at least 1999. 10,11,13dolase, speci cally muscle fructose-1,6-bisphosphate aldolase (ALDOA), provides a great example of glycolytic "moonlighting". 11ALDOA has the enzymatic function of catalyzing the reversible cleavage of fructose-1,6-bisphosphate to dihydroxyacetone-3-phosphate and glyceraldehyde-3-phosphate in glycolysis. 11However, its non-catalytic ability to organize actin laments, regulate Wnt and p53 signaling, help progression through the G1/S phase of the cell cycle have been described as its "moonlighting" ability.Thus, targeting these non-catalytic functions may present a novel anti-cancer therapy. 11Using the ALDOA slow-binding inhibitor UM0112176 in human non-small cell lung cancer cells, human pancreatic adenocarcinoma cells, rat astrocytes, and human epithelial cells, Gizak et al found that the pharmacological action of UM0112176 was independent of its inhibition of glycolysis and was speci c to cancer cells. 11Instead, it was observed that UM0112176 disrupted the interaction between ALDOA and actin. 11evious research on MCPyV-positive MCC cell lines has demonstrated that selinexor targets and decreases the expression of viral T antigens, 14 and inhibits the DNA damage response. 15Here, we describe the effects of selinexor on the expression of key glycolytic and metabolic enzymes and discuss both the direct catalytic effects on energy production and their indirect "moonlighting" effects.

Discussion
7][18] However, not all patients bene t from ICI or, especially those who are immunocompromised, are eligible to be treated with ICI. 19In addition, since half of MCCs are located on the head or neck rendering surgery di cult or impossible, then other therapies are required. 1he integration of MCPyV into the genome of MCC, speci cally the small T antigen, increase aerobic glycolysis among many other cellular changes. 9,20Therefore, targeting glycolytic genes to block this increased cellular respiration may be a viable treatment option.
Selinexor, a novel XPO1 inhibitor, is approved for patients with multiple myeloma. 21,22Selinexor was observed to decrease MCPyV-positive MCC cell proliferation by targeting the viral small T and large T antigens. 2,14,23,24Selinexor was previously tested on MCPyV-positive MCC cell lines MS-1 and MKL-1 using cell proliferation assays where the IC 50 value for MS-1 was determined to be 85.7 nmol/L. 15In addition, selinexor decreased the expression of DNA repair enzymes in MCC cells. 15Therefore, we hypothesized that selinexor may secondarily decrease glucose metabolism as a result of blocking the export of viral T antigens. 14ny tested genes including GLUD1, GLUT3, Hexokinase 1, and PFKFB2 demonstrated highly statistically signi cant downregulation of protein expression following selinexor treatment.Therefore, these genes may kill the MCC cells in this study in a manner like other reports of inhibition seen in other cancers.Due to the importance of glutaminolysis in cancer cells, several therapies have targeted this pathway. 25Although slightly distinct from the "Warburg effect", glutaminolysis was discovered to be necessary for many tumor cells because cells depended on glutamine for their survival and growth. 26utaminase converts glutamine into glutamate, which then can enter into the citric acid cycle. 26UD1 acts as a transaminase in the process of glutaminolysis by converting glutamate and ammonia into alpha-ketoglutaric acid. 27Breast, lung, and colorectal cancers have been reported to increase expression of GLUD1 during nutritional stress. 27In colorectal cancer, tumors with high levels of GLUD1 are often more aggressive, especially in times of nutritional stress. 27Glutaminase expression has been associated with decreased patient survival and lymph node metastasis in cancer. 28Some researchers propose the relationship between GLUD1 and glutaminase is through regulation of HIF-1α, a growth pathway upregulated in cancer, and the induction of the epithelial-mesenchymal transition. 28As a result, blocking glutaminase transcription may prevent MCC cancer aggression by decreasing glutaminolysis and other oncogenic cellular changes.
GLUT1 and GLUT3 are classical glucose transporters within the solute carrier membrane transport protein family. 29Beyond aiding in glucose transport and being associated with lower survival, GLUT3 transporters are reported to physically disturb other membrane-associated proteins, to increase gene expression through DNA demethylation and acetylation, and to co-regulate other proteins involved in glucose transport. 29Therefore, decreasing GLUT3 protein expression may also block downstream pathways.
Hexokinases have long been known to be the rst step of glycolysis, but its moonlighting functions, especially of hexokinase 1, are not fully understood. 30Since hexokinase 1 expression was downregulated in this study on MCC cells, selinexor may also be involved in decreasing cell proliferation through these other pathways.Hexokinases are known to act as intracellular glucose sensors and as regulators of apoptosis, autophagy, protein kinase expression, and transcription. 30,31Hexokinase 1 may increase the NADPH/NADP ratio in the absence of glucose as well as decrease the availability of amino acids. 30wever, more study is needed on the moonlighting role of hexokinase 1.
PFKFB2 is found localized to the nucleus in human pancreatic adenocarcinomas while it is primarily cytoplasmic in normal pancreatic cells. 32Transient PFKFB2 silencing in 2 types of pancreatic adenocarcinoma cell lines led to a modest and robust decrease in cell proliferation. 32Thus the nuclear PFKFB, may actually provide more non-metabolic functions and cellular proliferation than its cytoplasmic variant.PFKFB2 maintains high levels of fructose-2,6-bisphosphate in the cytoplasm and regulates glucose metabolism. 32linexor may decrease cell proliferation in MS-1 cell lines by blocking the expression of GLUD1, GLUT3, Hexokinase 1, and PFKFB2 since these genes are vital not only to the metabolic and energetic requirements but also in cell regulation as a whole.Protein levels of amphiregulin, LDHA, PDHK1, MCT1, G6PD, PKM2, and 6PGD were also found to be downregulated.These proteins also regulate a variety of non-metabolic functions that selinexor may indirectly target.Amphiregulin contributes to cancer proliferation through activating its receptor EGFR after cleavage and then functioning as a growth factor for several types of cells. 10Amphiregulin is known to facilitate glucose uptake via GLUT1 in colorectal cancers in addition to possibly enhancing the production of lactate. 10Lactate dehydrogenase catalytically (LDHA) converts pyruvate into lactate. 33In addition, LDHA may migrate to the nucleus to promote histone methylation in response to HPV16E7-induced reactive oxygen species (ROS) accumulation, activates antioxidant gene expression, and leads to cervical cancer cell proliferation under oxidative stress. 33Brighenti et al suggest that LDHA inhibitors such as GSK 2837808A which decrease cell proliferation work through hindering expression of the histone 2B gene rather than directly blocking aerobic glycolysis. 34Thus, a decrease in amphiregulin and LDHA expression may lead to decreased lactate production and prevent cell survival when under oxidative stress.
The monocarboxylate transporter (MCT) family, especially MCT1 and MCT4, is the primary exporter of lactate in tumors. 35However, MCT1 also seems to be non-catalytically involved in pyruvate export though the mechanism remains unclear. 356-phosphogluconate dehydrogenase (6PGD) is the third oxidative decarboxylase of the pentose phosphate pathway. 36While the non-catalytic mechanism by which 6PGD promotes cancer progression remains unclear, mediating lipogenesis, redox homeostasis, metastasis, and cell proliferation are all probable roles for 6PGD. 36Consequently, selinexor may also decrease pyruvate export, prevent lipogenesis, metastasis, and cell proliferation by targeting MCT1 and 6PGD.

Conclusion
In the MCC cell line MS-1, selinexor signi cantly downregulated expression of many genes in cellular energy metabolism and cellular proliferation in a statistically signi cant relevant manner.These data suggest that selinexor may be a viable treatment for MCC which is unresponsive to immune checkpoint inhibitors or to be used as a combination therapy.While the Warburg effect rst linked glycolysis and cancer to an increased energy demand and consumption, more recent research suggests the importance of the non-catalytic, moonlighting functions of these glycolytic enzymes.These other regulatory functions may provide the key to selinexor's e cacy in decreasing cell proliferation in MCC cell lines.
More studies in case reports and clinical trials are required to investigate these pathways and validate these ndings in case studies and blinded clinical trials.

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