Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of estrogen receptor, progesterone receptor, and the HER2 but is enriched with cancer stem celllike cells (CSC). CSCs are the fraction of cancer cells recognized as the source of primary malignant tumors that also give rise to metastatic recurrence. 5-Hydroxymethylcytosine (5hmC) is a DNA epigenetic feature derived from 5-methylcytosine by action of tet methylcytosine dioxygenase enzymes (e.g., TET1); and although TET1 and 5hmC are required to maintain embryonic stem cells, the mechanism and role in CSCs remain unknown. Data presented in this report support the conclusion that TET1 and TET1-dependent 5hmC mediate hydrogen peroxide (H 2 O 2 )-dependent activation of a novel gene expression cascade driving self-renewal and expansion of CSCs in TNBC. Evidence presented also supports that the H 2 O 2 affecting this pathway arises due to endogenous mechanismsincluding downregulation of antioxidant enzyme catalase in TNBC cells-and by exogenous routes, such as systemic inflammation and oxidative stress coupled with obesity, a known risk factor for TNBC incidence and recurrence.Implications: This study elucidates a pathway dependent on H 2 O 2 and linked to obesity-driven TNBC tumor-initiating CSCs; thus, it provides new understanding that may advance TNBC prevention and treatment strategies.

Obesity is a risk factor for triple‐negative breast cancer ( TNBC ) incidence and poor outcomes, but the underlying molecular biology remains unknown. We previously identified in TNBC cell cultures that expression of epigenetic reader methyl‐CpG‐binding domain protein 2 ( MBD 2), specifically the alternative mRNA splicing variant MBD variant 2 ( MBD 2_v2), is dependent on reactive oxygen species ( ROS ) and is crucial for maintenance and expansion of cancer stem cell‐like cells ( CSC s). Because obesity is coupled with inflammation and ROS , we hypothesized that obesity can fuel an increase in MBD 2_v2 expression to promote the tumor‐initiating CSC phenotype in TNBC cells in vivo . Analysis of TNBC patient datasets revealed associations between high tumor MBD 2_v2 expression and high relapse rates and high body mass index ( BMI ). Stable gene knockdown/overexpression methods were applied to TNBC cell lines to elucidate that MBD 2_v2 expression is governed by ROS ‐dependent expression of serine‐ and arginine‐rich splicing factor 2 ( SRSF 2). We employed a diet‐induced obesity ( DIO ) mouse model that mimics human obesity to investigate whether obesity causes increased MBD 2_v2 expression and increased tumor initiation capacity in inoculated TNBC cell lines. MBD 2_v2 and SRSF 2 levels were increased in TNBC cell line‐derived tumors that formed more frequently in DIO mice relative to tumors in lean control mice. Stable MBD 2_v2 overexpression increased the CSC fraction in culture and increased TNBC cell line tumor initiation capacity in vivo . SRSF 2 knockdown resulted in decreased MBD 2_v2 expression, decreased CSC s in TNBC cell cultures, and hindered tumor formation in vivo . This report describes evidence to support the conclusion that MBD 2_v2 expression is induced by obesity and drives TNBC cell tumorigenicity, and thus provides molecular insights into support of the epidemiological evidence that obesity is a risk factor for TNBC . The majority of TNBC patients are obese and rising obesity rates threaten to further increase the burden of obesity‐linked cancers, which reinforces the relevance of this report.

African American men (AAM) are at higher risk of being diagnosed with prostate cancer (PCa) and are at higher risk of dying from the disease compared to European American men (EAM). We sought to better understand PCa molecular diversity that may be underlying these disparities. We performed RNA‐sequencing analysis on high‐grade PCa to identify genes showing differential tumor versus noncancer adjacent tissue expression patterns unique to AAM or EAM. We observed that interleukin‐6 (IL‐6) was upregulated in the nonmalignant adjacent tissue in AAM, but in EAM IL‐6 expression was higher in PCa tissue. Enrichment analysis identified that genes linked to the function of TP53 were overrepresented and downregulated in PCa tissue from AAM. These RNA‐sequencing results informed our subsequent investigation of a diverse PCa cell line panel. We observed that PCa cell lines that are TP53 wild‐type, which includes cell lines derived from AAM (MDA‐PCa‐2b and RC77T), did not express detectable IL‐6 mRNA. IL‐6 treatment of these cells downregulated wild‐type TP53 protein and induced mRNA and protein expression of the epigenetic reader methyl CpG binding domain protein 2 (MBD2), specifically the alternative mRNA splicing variant MBD2_v2. Further investigation validated that upregulation of this short isoform promotes self‐renewal and expansion of PCa cancer stem‐like cells (CSCs). In conclusion, this report contributes to characterizing gene expression patterns in high‐grade PCa and adjacent noncancer tissues from EAM and AAM. The results we describe here advance what is known about the biology associated with PCa race disparities and the molecular signaling of CSCs.

Background Enhanced glucose metabolism is a feature of most tumors, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. Metabolic diseases including obesity and diabetes have a hyperglycemia component and are correlated with elevated pre-menopausal cancer risk for triple-negative breast cancer (TNBC). However, determining pathways for hyperglycemic disease-coupled cancer risk remains a major unmet need. One aspect of cellular sugar utilization is the addition of the glucose-derived protein modification O-GlcNAc (O-linked N-acetylglucosamine) via the single human enzyme that catalyzes this process, O-GlcNAc transferase (OGT). The data in this report implicate roles of OGT and O-GlcNAc within a pathway leading to cancer stem-like cell (CSC) expansion. CSCs are the minor fraction of tumor cells recognized as a source of tumors as well as fueling metastatic recurrence. The objective of this study was to identify a novel pathway for glucose-driven expansion of CSC as a potential molecular link between hyperglycemic conditions and CSC tumor risk factors. Methods We used chemical biology tools to track how a metabolite of glucose, GlcNAc, became linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-GlcNAc post-translational modification in three TNBC cell lines. Using biochemical approaches, genetic models, diet-induced obese animals, and chemical biology labeling, we evaluated the impact of hyperglycemia on CSC pathways driven by OGT in TNBC model systems. Results We showed that OGT levels were higher in TNBC cell lines compared to non-tumor breast cells, matching patient data. Our data identified that hyperglycemia drove O-GlcNAcylation of the protein TET1 via OGT-catalyzed activity. Suppression of pathway proteins by inhibition, RNA silencing, and overexpression confirmed a mechanism for glucose-driven CSC expansion via TET1-O-GlcNAc. Furthermore, activation of the pathway led to higher levels of OGT production via feed-forward regulation in hyperglycemic conditions. We showed that diet-induced obesity led to elevated tumor OGT expression and O-GlcNAc levels in mice compared to lean littermates, suggesting relevance of this pathway in an animal model of the hyperglycemic TNBC microenvironment. Conclusions Taken together, our data revealed a mechanism whereby hyperglycemic conditions activated a CSC pathway in TNBC models. This pathway can be potentially targeted to reduce hyperglycemia-driven breast cancer risk, for instance in metabolic diseases. Because pre-menopausal TNBC risk and mortality are correlated with metabolic diseases, our results could lead to new directions including OGT inhibition for mitigating hyperglycemia as a risk factor for TNBC tumorigenesis and progression.

3536 Background: Early-onset colorectal cancer (eoCRC, initial CRC diagnosis at age < 50 years) has been increasing in the past two decades especially in Western countries. This study evaluates somatic and germline profiles in eoCRC compared to average-onset CRC (aoCRC, initial CRC diagnosis at age ≥ 50 years). Methods: This is a retrospective, cross-sectional study utilizing data from de-identified records of colorectal cancer patients tested with the Tempus xT assay from 2017 to 2022. Briefly, the assay is a targeted panel that detects single nucleotide variants, insertions and/or deletions, and copy number variants in 598-648 genes, as well as chromosomal rearrangements in 22 genes with high sensitivity and specificity. Baseline characteristics and immunologic markers were compared between eoCRC and aoCRC by Wilcoxon Rank Sum or Chi-squared test (reporting p-values). Somatic and germline mutations were compared between two age groups with false discover rate adjustments (reporting q-values). Results: In this study, 2,379 eoCRC and 8,627 aoCRC patients were included, with the majority diagnosed with stage IV disease. Additionally, germline alterations were assessed on a subset of 6,311 patients with tumor/normal match testing (eoCRC = 1,413 and aoCRC = 4,898). Left-sided primaries were more common in eoCRC (85% left/rectum in eoCRC vs. 75% left/rectum in aoCRC (p < 0.001), and rates of microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) were lower compared to aoCRC (4.2% vs. 6.8%, p < 0.001). eoCRC has a unique somatic mutation profile compared to aoCRC. A higher prevalence of germline mutations was observed in eoCRC overall (6.9% vs. 5%, p = 0.006); however, no statistically significant differences were observed in individual germline genes compared to aoCRC, likely due to relatively small numbers. Conclusions: eoCRC has a unique mutational profile and presence of germline mutations in 6.9% of eoCRC, indicating a potential role for universal germline testing in CRC. [Table: see text]

Enhanced glucose metabolism is a feature of almost all cancers, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. The objective of this study is to characterize a mechanism by which elevated glucose level drives a tumorigenic pathway in triple negative breast cancer (TNBC). We used chemical biology methods to track how a metabolite of glucose, N-acetylglucosamine (GlcNAc), is linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-linked GlcNAc post translational modification (O-GlcNAc). In this work, we revealed that intracellular protein glycosylation by O-GlcNAc is driven by high glucose levels in TNBC models, including on TET1. A single enzyme, O-GlcNAc transferase (OGT), is responsible for catalyzing protein modification of O-GlcNAc. We showed that OGT activity is higher in TNBC cell lines compared to non-tumor breast cell lines and is associated with hyperglycemia. Furthermore, enhanced OGT activity activated a pathway for cancer stem-like cell (CSC) reprogramming in TNBC cells. In our model, O-GlcNAcylated TET1 upregulated expression of splicing factor TAR-DNA binding protein (TARDBP), which drives CSC induction as well as higher OGT levels. We show that this OGT-TET1-TARDBP axis 'feeds-forward' in hyperglycemic conditions both in cell lines and diet-induced obese mice, which displayed higher blood glucose levels and tumor O-GlcNAc levels than lean littermates. This data converges on a novel pathway whereby hyperglycemia drives aberrant OGT activity, activating a pathway for CSC induction in TNBC. Our findings partially explain a key aspect of how obesity is associated with TNBC risk and negative outcomes.

<p>Supplementary Data Figures S1-S4</p>

<p>Supplementary Data Figures S1-S4</p>