O-linked-N-acetylglucosaminylation (O-GlcNAcylation), a dynamic post-translational modification (PTM), holds profound implications in controlling various cellular processes such as cell signaling, metabolism, and epigenetic regulation that influence cancer progression and therapeutic resistance. From the therapeutic perspective, O-GlcNAc modulates drug efflux, targeting and metabolism. By integrating signals from glucose, lipid, amino acid, and nucleotide metabolic pathways, O-GlcNAc acts as a nutrient sensor and transmits signals to exerts its function on genome stability, epithelial-mesenchymal transition (EMT), cell stemness, cell apoptosis, autophagy, cell cycle. O-GlcNAc also attends to tumor microenvironment (TME) and the immune response. At present, several strategies aiming at targeting O-GlcNAcylation are under mostly preclinical evaluation, where the newly developed O-GlcNAcylation inhibitors markedly enhance therapeutic efficacy. Here we systematically outline the mechanisms through which O-GlcNAcylation influences therapy resistance and deliberate on the prospects and challenges associated with targeting O-GlcNAcylation in future cancer treatments. 

Targeting O-GlcNAcylation in cancer therapeutic resistance: The sugar Saga continues.

Gene transcription is a highly regulated process, and deregulation of transcription factors activity underlies numerous pathologies including cancer. Albeit near four decades of studies have established that the E2F pathway is a core transcriptional network that govern cell division in multi-cellular organisms1,2, the molecular mechanisms that underlie the functions of E2F transcription factors remain incompletely understood. FOXK1 and FOXK2 transcription factors have recently emerged as important regulators of cell metabolism, autophagy and cell differentiation3–6. While both FOXK1 and FOXK2 interact with the histone H2AK119ub deubiquitinase BAP1 and possess many overlapping functions in normal biology, their specific functions as well as deregulation of their transcriptional activity in cancer is less clear and sometimes contradictory7–13. Here, we show that elevated expression of FOXK1, but not FOXK2, in primary normal cells promotes transcription of E2F target genes associated with increased proliferation and delayed entry into cellular senescence. FOXK1 expressing cells are highly prone to cellular transformation revealing important oncogenic properties of FOXK1 in tumor initiation. High expression of FOXK1 in patient tumors is also highly correlated with E2F gene expression. Mechanistically, we demonstrate that FOXK1, but not FOXK2, is specifically modified by O-GlcNAcylation. FOXK1 O-GlcNAcylation is modulated during the cell cycle with the highest levels occurring during the time of E2F pathway activation at G1/S. Moreover, loss of FOXK1 O-GlcNAcylation impairs FOXK1 ability to promote cell proliferation, cellular transformation and tumor growth. Mechanistically, expression of FOXK1 O-GlcNAcylation-defective mutants results in reduced recruitment of BAP1 to gene regulatory regions. This event is associated with a concomitant increase in the levels of histone H2AK119ub and a decrease in the levels of H3K4me1, resulting in a transcriptional repressive chromatin environment. Our results define an essential role of O-GlcNAcylation in modulating the functions of FOXK1 in controlling the cell cycle of normal and cancer cells through orchestration of the E2F pathway.

O-GlcNAcylation of FOXK1 orchestrates the E2F pathway and promotes oncogenesis

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 μg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.

Studying the O-GlcNAcome of human placentas using banked tissue samples.

O-GlcNAc informatics: advances and trends

The relationship between O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and mitosis is intertwined. Besides the numerous mitotic OGT substrates that have been identified, OGT itself is also a target of the mitotic machinery. Previously, our investigations have shown that Checkpoint kinase 1 (Chk1) phosphorylates OGT at Ser-20 to increase OGT levels during cytokinesis, suggesting that OGT levels oscillate as mitosis progresses. Herein we studied its underlying mechanism. We set out from an R17C mutation of OGT, which is a uterine carcinoma mutation in The Cancer Genome Atlas. We found that R17C abolishes S20 phosphorylation of OGT, as it lies in the Chk1 phosphorylating consensus motif. Consistent with our previous report that pSer-20 is essential for OGT level increases during cytokinesis, we further demonstrate that the R17C mutation renders OGT less stable, decreases vimentin phosphorylation levels and results in cytokinesis defects. Based on bioinformatic predictions, pSer-20 renders OGT more likely to interact with 14-3-3 proteins, the phospho-binding signal adaptor/scaffold protein family. By screening the 7 isoforms of 14-3-3 family, we show that 14-3-3ε specifically associates with Ser-20-phosphorylated OGT. Moreover, we studied the R17C and S20A mutations in xenograft models and demonstrated that they both inhibit uterine carcinoma compared to wild-type OGT, probably due to less cellular reproduction. Our work is a sequel of our previous report on pS20 of OGT and is in line with the notion that OGT is intricately regulated by the mitotic network. 

14-3-3ε augments OGT stability by binding with S20-phosphorylated OGT

Cell cycle errors can lead to mutations, chromosomal instability, or death; thus, the precise control of cell cycle progression is essential for viability. The nutrient-sensing post-translational modification, O-GlcNAc, regulates the cell cycle allowing one central control point directing progression of the cell cycle. O-GlcNAc is a single N-acetylglucosamine sugar modification to intracellular proteins that is dynamically added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. These enzymes act as a rheostat to fine-tune protein function in response to a plethora of stimuli from nutrients to hormones. O-GlcNAc modulates mitogenic growth signaling, senses nutrient flux through the hexosamine biosynthetic pathway, and coordinates with other nutrient-sensing enzymes to progress cells through Gap phase 1 (G1). At the G1/S transition, O-GlcNAc modulates checkpoint control, while in S Phase O-GlcNAcylation coordinates the replication fork. DNA replication errors activate O-GlcNAcylation to control the function of the tumor suppressor p53 at Gap Phase 2 (G2). Finally, in Mitosis (M phase), O-GlcNAc controls M phase progression and the organization of the mitotic spindle and midbody. Critical for M phase control is the interplay between OGT and OGA with mitotic kinases. Importantly, disruptions in OGT and OGA activity induce M phase defects and aneuploidy. These data point to an essential role for the O-GlcNAc rheostat in regulating cell division. In this review, we highlight O-GlcNAc nutrient sensing regulating G1; O-GlcNAc control of DNA replication and repair; and finally, O-GlcNAc organization of mitotic progression and spindle dynamics. 

Growing and dividing: how O-GlcNAcylation leads the way.


 We previously reported that collagen type XI alpha 1 (COL11A1) is upregulated in head and neck squamous cell carcinoma (HNSCC) and contributes to tumor progression. Here we tested the hypothesis that COL11A1 regulates the expression of immune regulatory markers in HNSCC. Xena Browser was used to analyze 520 and 42 cancer free normal head and neck tissue samples for COL11A1 expression. The Cancer Genome Atlas (TCGA) RNASeq data from 522 HNSCC samples was downloaded and sorted by clinical parameters and RNA expression for 20,000 genes. COL11A1 expression was correlated with tumor stage (T) and clinical stage (I-IV). Data was sorted by COL11A1 high and low based on the mean COL11A1 expression level and run through the CIBERSORT analysis tool for prediction of immune cell proportions in the tumor. HNSCC lines with COL11A1 knockdown were assessed for immune regulatory markers at the RNA and protein level. COL11A1 was found to be increased in HNSCC relative to normal tissue (tumor vs. normal p<0.0001). COL11A1 expression is highest in HPV negative HNSCC. Its expression correlates with increased tumor (T1 vs T4 p=0.033) and clinical stage (stage III vs. stage IV p=0.023). CIBERSORT analysis demonstrates COL11A1hi patients have significantly decreased active natural killer (NK) cells (active vs. inactive p=0.0166), CD8+ T cells (CD8 low vs. high p<0.0001) and active dendritic cells (active vs. inactive p=0.0035) suggesting immune modulation. COL11A1 targeted lines in vitro show decreased expression of hla-a, -c and cd274 (PD-L1), suggesting COL11A1 expression modulates immune activation by regulating immune suppressive ligands on the tumor cell. COL11A1 is overexpressed in HNSCC and drives tumor proliferation, migration and invasion. Expression levels correlate with tumor and clinical stage. Bioinformatics tool CIBERSORT, predicts decreased immune cell activity in patients with high COL11A1. Targeting COL11A1 in vitro decreased expression of immune suppressive ligands. These preliminary results indicate COL11A1 may drive immune evasion in HPV negative HNSCC. Targeting COL11A1 may improve patient response to immunotherapeutic targets in HNSCC.
 Citation Format: Harmony Ivanna Saunders, Levi Arnold, Mary Markiewicz, Sufi M. Thomas. COL11A1: A driver of tumor progression and immune evasion in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3844.

Abstract 3844: COL11A1: A driver of tumor progression and immune evasion in head and neck squamous cell carcinoma (HNSCC)


 The role of hormone receptors in breast cancer has been well established as a key contributor to breast cancer tumorigenesis. Estrogen receptor and progesterone receptors are present in nearly 70% of breast cancers. The role of estrogen receptor and estrogens in breast cancer has been extensively studied and targeted effectively through endocrine therapy, however the impact of progesterone and the progesterone receptor (PR) independent of estrogen is not well understood. We have previously demonstrated that PR promotes tumor growth and drives an immune suppressive environment, therefore understanding the biology of PR in breast cancer is critical. We have also demonstrated that PR attenuates type 1 interferon signaling via inhibition of STAT1 phosphorylation and increased degradation of STAT2. Post-translational modifications such as phosphorylation and SUMOylation can influence PR target gene promoter selectivity thereby altering its function. Our recent work indicates PR is modified by O-GlcNAc, a single N-acetyl-glucosamine sugar that cycles on and off and serine or threonine amino acids in nuclear, cytoplasmic and mitochondrial proteins. Levels of total O-GlcNAc staining are higher in breast cancer tissue compared to adjacent normal tissue. Active O-GlcNAcylation, as evidenced by immunohistochemistry staining of O-GlcNAc-transferase (OGT), is elevated in patients with PR+ tumors compared to PR-. Using T47D breast cancer cells (an ER/PR-positive tumor line), mass spectrometry analysis revealed an O-GlcNAc site at S499 on PR. We then used a naturally occurring PR-negative variant of T47D cells to introduce stable expression of a mutant PR with serine to alanine substitution at the O-GlcNAc site (S499A), thereby blocking O-GlcNAc flux, in addition to wt PR as a control. RNA-Seq analysis of these cells following treatment with progesterone revealed several significant differential gene expression patterns, including rescue of PR dependent interferon signaling attenuation. T47D-mutant PR cell lines implanted into immune deficient mice had significantly decreased growth as compared to WT-PR control tumors. In syngeneic mice harboring PR-positive and PR-negative tumors, loss of O-GlcNAc flux and increased O-GlcNac levels increased tumor growth only when tumors were expressing PR, corroborating the influence of O-GlcNAc in immunodeficient mice. Together these findings indicate O-GlcNAcylation of PR is potentially a mechanism of PR driven breast cancer tumorigenesis and immune evasion.
 Citation Format: Harmony Ivanna Saunders, Sean M. Holloran, Gloria Trinca, Chad Slawson, Christy Hagan. O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5280.

Abstract 5280: O-GlcNAcylation of progesterone receptor promotes mammary tumorigenesis


 Breast Cancer is the most common cancer diagnosed in women in the United States. Second only to lung cancer, breast cancer results in the greatest number of cancer deaths for women. Hormone receptor positive (HR+) breast cancers - cancers expressing the nuclear receptors for steroid hormones estrogen and progesterone, termed the estrogen receptor (ER) and progesterone receptor (PR) - make up the largest proportion of diagnosed breast cancers by a significant margin. So, although the HR+ subtype is less aggressive compared to others, it continues to be the largest contributor to breast cancer mortality. Treatment consists of anti-endocrine therapies, largely targeting ER/estrogen, the established drivers of breast cancer growth. Most women respond well to these therapies, but for women who do not, there are few alternative options. With the advent of immunotherapy and its successes in treating and controlling certain tumor types, many were hopeful that it could be employed for breast cancer as well. However, breast cancers, especially HR+ breast cancers, are considered “immune cold.” They lack high proportions of tumor-infiltrating lymphocytes (TILs), have low neoantigen production, and high percentages of immunosuppressive cells. A contributing factor could be the hormones present in the tumor microenvironment. The accepted paradigm is that lifetime exposure to estrogens drives the development of HR+ breast cancer, but both estrogen and progesterone are part of the normal menstrual cycle. Prior work from our lab has uncovered a novel immune-modulatory role for progesterone, pivotal to promoting the growth of PR-positive mammary tumors. Recent preliminary data from our lab show that progesterone treatment conveys immune protection against T cell killing in a co-culture with E0771 mammary gland tumor cells. However, when activated in monoculture, T cells exposed to increasing concentrations of progesterone have no differing expression of cell surface activation or exhaustion markers, implying that progesterone is not eliciting this effect by interfering with the T cell activation cascade nor is it inducing premature exhaustion. Interestingly, progesterone treatment instead leads to a concentration-dependent increase in T cell death compared to the untreated control. In vivo, these results are supported as progesterone treatment decreases the amount of total CD3+ T cells, and specifically the cytotoxic CD8+ subset, in the mammary glands of mice. These results suggest that progesterone could modulate the breast and tumor microenvironment, providing tumor cells with protection from immunosurveillance and contributing to HR+ breast cancer development. These data also argue for the use of anti-progestins to be added to the HR+ breast cancer treatment regimen to boost anti-tumor immune activity.
 Citation Format: Amanda Heard, Lauryn R. Werner, Eilidh I. Chowanec, Harmony Saunders, Julio Tinoco, Zachary C. Hartman, Christy R. Hagan. Progesterone promotes immunosuppression in the murine mammary gland by causing T cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6877.

Abstract 6877: Progesterone promotes immunosuppression in the murine mammary gland by causing T cell death


 Hormone receptor (HR) positive breast cancers, which account for approximately 75% of all breast cancers and express both estrogen receptor (ER) and progesterone receptor (PR), have been the subject of much research. As such, successful anti-estrogen/ER-based therapies have been very successful in the treatment of HR+ breast cancer. Despite this success, over 1/3 of patients will eventually progress on current ER/estrogen-targeted therapies, underscoring the need for new targeted therapies in HR+ breast cancer. Unlike ER, the role of PR in breast cancer progression has received far less attention. In a previous study, we found that a mouse mammary tumor cell line E0771, modified to express the mouse progesterone receptor (mPR) and the OVA peptide, demonstrated decreased T cell-mediated cell death when treated with progesterone compared to the control group. Progesterone-mediated protection from T-cell death was only observed in mPR+ cell lines. Flow analysis of the E0771-Ova-mPR cells also revealed a decrease in MHC Class I presentation in progesterone-treated cells compared to a vehicle control. To further explore the role of the machinery required for antigen processing and presentation of MHC Class I molecules, we conducted additional experiments using the E0771 cell line. Our results showed that treatment with progesterone led to a reduction in RNA levels of all the machinery required for antigen processing and presentation (APP). Based on these findings, we hypothesize that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by regulating APP. Our results showed that treatment with progesterone resulted in a reduction in RNA levels of Tap1, Tap2, Tapbp, Nlrc5, B2m, and Psmb8, which are essential for APP. We therefore propose that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by directly modulating the APP pathway. The findings of this study may have significant implications for our understanding of how breast cancer cells evade T-cell mediated cytotoxicity.
 Citation Format: Julio Tinoco, Lauryn Werner, Eilidh Chowanec, Harmony Saunders, Sun Xiaopeng, Jay Gertz, Justin Balko, Zachary C. Hartman, Christy Hagan. Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5364.

Abstract 5364: Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors

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