Translational potential of SENP1 in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal cancer with an estimated 5-year survival rate of <20% (1). In addition to late-stage diagnosis and frequent metastasis, therapeutic resistance remains a significant challenge in ESCC (1). Despite much interest, the development of effective targeted therapies for ESCC remains elusive.

SUMOylation is a reversible post-translational modification through which small ubiquitin-like modifier (SUMO) moieties are covalently linked to lysine residues in target proteins. SUMOylation may influence protein stability, localization, and interactions, affecting diverse cellular processes, including gene expression, DNA repair, and signal transduction (2,3). The SUMO pathway influences the stability and function of oncogenes and tumor suppressors to influence carcinogenesis (4). Sentrin-specific proteases (SENPs) are enzymes that catalyze the removal of SUMO from their substrates. SENP1 upregulation has been associated with poor outcomes in many cancers, including acute myeloid leukemia (5), prostate cancer (6), Wilms tumor (7), renal cell carcinoma (8), nasopharyngeal carcinoma (9), and colorectal cancer (10). Mechanisms through which SENP1 contributes to carcinogenesis include promoting proliferation, angiogenesis, invasion, metastasis, inhibiting apoptosis, and altering cellular metabolism (11). Various agents have been shown to inhibit SENP1. The natural product triptolide inhibits SENP1 expression and exerts anti-tumor properties in preclinical cancer models of prostate cancer (12). A phase I clinical trial of minnelide, a water-soluble prodrug of triptolide, in patients with refractory gastrointestinal cancers recently established safety of this agent while also providing clinical evidence of anti-tumor activity (13).

Our understanding of the SUMO pathway in ESCC remains limited. SUMOylation of specific proteins, including heat shock protein (HSP27) and mini chromosome maintenance protein 10 (MCM10), has been linked to ESCC progression (14,15). Additionally, three recent studies explored SENP1, SENP2, and SENP3 individually in ESCC (16-18). Among these reports, Gu et al. focused on SENP1 (16), evaluating ESCC data from The Cancer Genome Atlas (TCGA) database to reveal increased SENP1 expression in tumors compared to normal adjacent mucosa. SENP1 upregulation was also documented at the level of RNA and protein in both a cohort of human patients from China and mice treated with the oral-esophageal carcinogen 4-nitroquinoline 1-oxide (4NQO). In human subjects, SENP1 expression further correlated with primary tumor stage and poor prognosis. The authors continued to investigate the functional significance of SENP1 in ESCC, performing in vitro studies with genetic SENP1 depletion. These studies revealed that SENP1 promotes proliferation and migration of ESCC cell lines. To gain insight into the effects of SENP1 depletion in vivo, the authors first performed xenograft experiments with SENP1-depleted ESCCs, which generated smaller tumors than the tumors expressing a non-targeting short hairpin RNA (shRNA). The authors also crossed Senp1^loxP/loxP mice with ED-L2-Cre mice, a strategy that facilitates Senpi1 conditional knockout (cKO) in the esophagus, forestomach, and ventral neck as Cre recombinase expression is under the control of the Epstein-Barr virus (EBV) L2 promoter. After treatment with 4NQO, Senpi1 cKO mice displayed a decrease in tumor number and intratumoral Ki67 density as compared to control mice lacking Cre. These in vitro and in vivo studies support SENP1 as a novel promoter of malignant features of ESCC cells.

Gu et al. next sought to explore the mechanisms through which SENP1 contributes to ESCC progression. RNA-sequencing and gene ontology analysis in KYSE150 ESCC cells revealed that genetic depletion of SENP1 was associated with alterations in the cell cycle pathway, including decreased expression of genes encoding cyclin A, cyclin D, thymidine kinase 1, geminin, and cyclin-dependent kinase 1, all of which promote cell proliferation. A positive relationship between SENP1 and the cell cycle was validated as SENP1 depletion in synchronized KYSE150 cells resulted in accumulation of cells in G1 phase of the cell cycle concomitant with diminished representation of cells in G2 phase and mitosis. The authors further reported no significant difference in apoptosis in SENP1-depleted KYSE150 cells as compared to their counterparts expressing non-targeting shRNA.

Gu et al. continued to evaluate the deacetylase and mono-ADP ribosyltransferase sirtuin (SIRT) 6 as a potential mediator through which SENP1 promotes proliferation in ESCC cells. The justification for the selection of SIRT6 as a candidate mediator of this effect is based upon SIRT6 serving as a tumor suppressor in multiple cancers and the authors’ previous report that SUMOylation modulates the tumor suppressive activities of SIRT6 (19). It is worth noting that SIRT6, has been shown to be upregulated in human ESCC tumors and to promote proliferation, autophagy, and expression of Bcl2 in ESCC cells in vitro (20). Gu et al. reported that SIRT6 depletion decreased proliferation in SENP1-depleted ESCC KYSE150 cells. Co-immunoprecipitation experiments then demonstrated that levels of SUMO-conjugated SIRT6 are increased upon SENP1 depletion, suggesting that SENP1 deSUMOylates SIRT6. Previously, this group reported that SIRT6 SUMOylation deficiency reduces SIRT6-mediated deacetylation of histone H3 at lysine 56 (H3K56) and promotes SIRT6-mediated repression of c-Myc target genes in HEK293 cells and mouse embryonic fibroblasts (MEFs) (19). Additionally, SIRT6 SUMOylation deficiency limited the tumor suppressor activity of SIRT6 in both lung and adenocarcinoma cell lines (19). As such, Gu et al. explored the relationship between SENP1, SIRT6, and H3K56 acetylation in KYSE150 cells. These studies revealed that SENP1 depletion reduced H3K56 acetylation, and this effect was rescued upon genetic depletion of SIRT6. In the context of dual depletion of SENP1 and SIRT6, reintroduction of wild type SIRT6 decreased H3K56 acetylation and cell proliferation; however, reintroduction of a SUMOylation-deficient SIRT6 mutant failed to significantly influence either H3K56 acetylation or cell proliferation. Thus, the authors propose a novel mechanism whereby SENP1 promotes ESCC growth by decreasing SUMOylation of tumor suppressor SIRT6.

The study by Gu et al. identifies SENP1 as a potential therapeutic target in ESCC. Based on TCGA data analysis in the current study, it appears that a subset of ESCC patients may fail to exhibit upregulation of SENP1 at the level of RNA. The demonstrated paired analyses of normal and tumor tissues from ESCC patients further suggest that SENP1 expression may indeed be increased in ESCC lesions; however, these studies should be expanded beyond the limited sample size utilized by Gu et al. As the current study found that high expression of SENP1 is associated with advanced primary tumor stage and poor prognosis, further exploration of SENP1 as a biomarker in ESCC is warranted. To complement the in vitro studies by Gu et al. linking SENP1 depletion to SIRT6-mediated deacetylation of H3K56, expression of SENP1 in ESCC lesions could be correlated to levels of SIRT6 and acetylated H3K56 using immunostaining. Senpi1 cKO mice would also be expected to display increased SIRT6-mediated H3K56 deacetylation. As Sirt6 floxed mice are available, it would be interesting to determine if the impaired tumor growth in response to 4NQO that is observed in Senpi1 cKO mice is restored in Senpi1;Sirt6 double cKO mice. It further remains to be determined whether SENP1 depletion results in repression of c-Myc target genes in ESCC cells as was observed in HEK293 cells (19). Should SENP1 inhibition limit expression of c-Myc target genes in ESCC, these findings would support prioritizing testing of SENP1 inhibition in the 23% of ESCC patients in which MYC is amplified (21).

Genomic analysis of ESCC revealed three molecular subtypes: ESCC1, ESCC2, and ESCC3 (21). ESCC1 is common in the Asian population and corresponds with activation of the NRF2-oxidative stress pathway; ESCC2 is common in individuals from Eastern Europe and South America and is associated with a high level of NOTCH1 mutation. ESCC3 is identified in individuals from the United States and Canada, and features PI3K signaling alterations without cell cycle gene changes. Given this molecular heterogeneity, consideration of whether increased SENP1 expression is unique to a specific molecular subtype of the disease may help to inform the potential utility of SENP1 as a biomarker and a therapeutic target in ESCC. ESCC1 shows a higher rate of YAP1 amplification as compared to ESCC2 and ESCC3 (21). In hepatic stellate cells, transcriptional co-activator YAP1 and transcription factor TAZ, key regulators of Hippo signaling, have been shown to be deacetylated by SIRT6 leading to their inhibition (22). Should SENP1 be upregulated in ESCC1, it is plausible that SIRT6 activity may be diminished, thereby limiting deacetylation of YAP and TAZ and promoting Hippo signaling. In embryonic stem cells, SIRT6 deacetylates the SOX2 promoter at H3K56 and H3K9 to repress SOX2 expression (23). As ESCC1 and ESCC2 exhibit amplification of SOX2 (21), SENP1 upregulation in these ESCC subtypes may limit SIRT6 activity, thereby promoting SOX2 expression. Although the described literature provides potential mechanisms through which SENP1 may contribute to the molecular heterogeneity of ESCC, the direct relationship between SENP1 expression and molecular subtypes of ESCC has yet to be established.

Notably, the current study by Gu et al. did not employ SENP1 inhibitors. Thus, studies evaluating agents shown to inhibit SENP1 expression, should be performed in ESCC preclinical models. A recent report demonstrated that triptolide, which inhibits SENP1 expression in prostate cancer cells (12), induces apoptosis while limiting proliferation, migration, and invasion of ESCC cells in vitro (24). This study, which also found that triptolide limited ESCC xenograft tumor growth, linked the effects of triptolide to the regulation of a NOX4 circular RNA-related pathway (24). It is possible, however, that SENP1-mediated SUMOylation of SIRT6 may also contribute to the anti-tumor effects of triptolide in ESCC. It is important to note that SENP1 is widely distributed across mammalian tissues (testes, ovaries, pancreas, etc.) (25), and thus proper dosage and targeted drug delivery methods will most likely play a crucial role in limiting patient toxicities and off-target effects. As SENP2 and SENP3 are also upregulated in ESCC cells where they promote malignant properties (17,18), broad targeting of SENPs may also be tested in ESCC.

While the current study by Gu et al. as well as the noted studies exploring SENP2 and SENP3 in ESCC focus on the role of these proteases in epithelial cells, roles for SUMOylation in inflammation and anticancer immunity have been reported. Inhibition of SUMOylation has been reported to enhance the antitumor immune response through mechanisms that include upregulation of interferon (INF)-β and IFN-1 to activate innate immune cells, including natural killer (NK) cells and macrophages (26). SUMOylation is also crucial for regulatory T cell (T reg) activity and maintenance (27,28). In lymphoma cells, pharmacologic inhibition of SUMOylation increased major histocompatibility complex I (MHC-I) expression on tumor cells and this effect was further augment by IFN-γ (29). Thus, inhibition of SUMOylation has the potential to promote an anti-tumor inflammatory milieu while simultaneously driving T cell recognition signals in cancer cells. Presently, SUMOylation inhibitors, including TAK981, are in clinical trials in both hematological malignancies and solid tumors (30,31). Preclinical studies using a syngeneic colorectal cancer xenograft model demonstrated that TAK981 potentiated immune checkpoint blockade therapy (32), further supporting the potential of targeting SUMOylation as an approach to improving cancer immunotherapy response. With regard to ESCC, SENP3 has been shown to be upregulated in macrophages in the tumor microenvironment where it limits disease progression by influencing macrophage polarization (33). Additionally, Gu et al. demonstrate that SENP1 depletion impairs tumor proliferation without impacting apoptosis, the latter of which may generate neoantigens to contribute to an anti-tumor response. These findings underscore the need for further investigation of the SUMO cascade, including specific SUMOylation targets (e.g., SIRTs) and modulators (e.g., SENPs) in human malignancies in a disease- and context-dependent manner. Ultimately, exploration of SENP1 inhibitors in the context of ESCC holds promise for enhancing treatment strategies, but a comprehensive evaluation of their efficacy, safety, and potential for clinical application remains crucial to advancing patient care and outcomes.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Esophagus. The article has undergone external peer review.

Peer Review File: Available at https://aoe.amegroups.com/article/view/10.21037/aoe-24-28/prf

Funding: This work was supported by a Bridge award, an internal grant provided by the Office of the Vice President for Research (OVPR) at Temple University.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://aoe.amegroups.com/article/view/10.21037/aoe-24-28/coif). K.A.W. was supported by NIH and NDDK and received a grant from Department of Defense, Laurel Harvest Labs, NIH and NIDCR. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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