Racial disparities in the clinical outcomes of triple-negative breast cancer (TNBC) have been well-documented, but the underlying biological mechanisms remain poorly understood. To investigate these disparities, we employed a multi-omic approach integrating imaging mass cytometry and spatial transcriptomics to characterize the tumor microenvironment (TME) in self-identified Black American (BA) and White American (WA) TNBC patients. Our analysis revealed that the TME in BA patients is marked by a network of endothelial cells, macrophages, and mesenchymal-like cells, which correlates with reduced patient survival. In contrast, the WA TNBC microenvironment is enriched in T-cells and neutrophils, indicative of T-cell exhaustion and suppressed immune responses. Ligand-receptor and pathway analyses further demonstrated that BA TNBC tumors exhibit a relatively "immune-cold" profile, while WA TNBC tumors display features of an "inflamed" TME, suggesting the evolution of a unique immunosuppressive mechanism. These findings provide insight into racially distinct tumor-promoting and immunosuppressive microenvironments, which may contribute to the observed differences in clinical outcomes among BA and WA TNBC patients.

Supplementary Material and Methods

Bladder cancer (BLCA) mortality is higher in African American (AA) patients compared with European American (EA) patients, but the molecular mechanism underlying race-specific differences are unknown. To address this gap, we conducted comprehensive RNA-Seq, proteomics, and metabolomics analysis of BLCA tumors from AA and EA. Our findings reveal a distinct metabolic phenotype in AA BLCA characterized by elevated mitochondrial oxidative phosphorylation (OXPHOS), particularly through the activation of complex I. The results provide insight into the complex I activation-driven higher OXPHOS activity resulting in glutaminemediated metabolic rewiring and increased disease progression, which was also confirmed by [U]13C-glutamine tracing. Mechanistic studies further demonstrate that knockdown of NDUFB8, one of the components of complex I in AA BLCA cells, resulted in reduced basal respiration, ATP production, GLS1 expression, and proliferation. Moreover, preclinical studies demonstrate the therapeutic potential of targeting complex I, as evidenced by decreased tumor growth in NDUFB8depleted AA BLCA tumors. Additionally, genetic and pharmacological inhibition of GLS1 attenuated mitochondrial respiration rates and tumor growth potential in AA BLCA. Taken together, these findings provide insight into BLCA disparity for targeting GLS1-Complex I for future therapy.

Abstract Approximately one-third of endocrine-treated women with estrogen receptor alpha–positive (ER+) breast cancers are at risk of recurrence due to intrinsic or acquired resistance. Thus, it is vital to understand the mechanisms underlying endocrine therapy resistance in ER+ breast cancer to improve patient treatment. Mitochondrial fatty acid β-oxidation (FAO) has been shown to be a major metabolic pathway in triple-negative breast cancer (TNBC) that can activate Src signaling. Here, we found metabolic reprogramming that increases FAO in ER+ breast cancer as a mechanism of resistance to endocrine therapy. A metabolically relevant, integrated gene signature was derived from transcriptomic, metabolomic, and lipidomic analyses in TNBC cells following inhibition of the FAO rate-limiting enzyme carnitine palmitoyl transferase 1 (CPT1), and this TNBC-derived signature was significantly associated with endocrine resistance in patients with ER+ breast cancer. Molecular, genetic, and metabolomic experiments identified activation of AMPK-FAO-oxidative phosphorylation (OXPHOS) signaling in endocrine-resistant ER+ breast cancer. CPT1 knockdown or treatment with FAO inhibitors in vitro and in vivo significantly enhanced the response of ER+ breast cancer cells to endocrine therapy. Consistent with the previous findings in TNBC, endocrine therapy–induced FAO activated the Src pathway in ER+ breast cancer. Src inhibitors suppressed the growth of endocrine-resistant tumors, and the efficacy could be further enhanced by metabolic priming with CPT1 inhibition. Collectively, this study developed and applied a TNBC-derived signature to reveal that metabolic reprogramming to FAO activates the Src pathway to drive endocrine resistance in ER+ breast cancer. Significance: Increased fatty acid oxidation induced by endocrine therapy activates Src signaling to promote endocrine resistance in breast cancer, which can be overcome using clinically approved therapies targeting FAO and Src.

SMARCB1 loss has long been observed in many solid tumors. However, there is a need to elucidate targetable pathways driving growth and metastasis in SMARCB1-deficient tumors. Here, we demonstrate that SMARCB1 deficiency, defined as genomic SMARCB1 copy number loss associated with reduced mRNA, drives disease progression in patients with bladder cancer by engaging STAT3. SMARCB1 loss increases the chromatin accessibility of the STAT3 locus in vitro. Orthotopically implanted SMARCB1 knockout (KO) cell lines exhibit increased tumor growth and metastasis. SMARCB1-deficient tumors show an increased IL6/JAK/STAT3 signaling axis in in vivo models and patients. Furthermore, a pSTAT3 selective inhibitor, TTI-101, reduces tumor growth in SMARCB1 KO orthotopic cell line-derived xenografts and a SMARCB1-deficient patient derived xenograft model. We have identified a gene signature generated from SMARCB1 KO tumors that predicts SMARCB1 deficiency in patients. Overall, these findings support the clinical evaluation of STAT3 inhibitors for the treatment of SMARCB1-deficient bladder cancer.

Supplementary Figure S4. A) Survival analysis of the upregulated (11/27) gene signature in high grade BLCA from the Kim, Lindgren, Sjodahl, and TCGA coherts. B) Survival analysis of the upregulated (11/27) gene signature in low grade BLCA from the Kim, Lindgren, and Sjodahl coherts. Upregulated genes associated with poor survival in the TCGA high-grade (log-rank p<0.0041), the Lindgren high grade cohort (log-rank p<0.0298), and in the Sjodahl low-grade cohort (log-rank p<0.0418). (LG=low-grade, HG= high-grade).

Supplementary Table S4. List of 519 differential metabolites, m/z, log fold change, and linear fold change.

BACKGROUND:Hypertension is the largest risk factor affecting global mortality. Despite available medications, uncontrolled hypertension is on the rise, whereby there is an urgent need to develop novel and sustainable therapeutics. Because gut microbiota is now recognized as an important entity in blood pressure regulation, one such new avenue is to target the gut-liver axis wherein metabolites are transacted via host-microbiota interactions. Knowledge on which metabolites within the gut-liver axis regulate blood pressure is largely unknown.METHOD:To address this, we analyzed bile acid profiles of human, hypertensive and germ-free rat models and report that conjugated bile acids are inversely correlated with blood pressure in humans and rats.RESULTS:Notably intervening with taurine or tauro-cholic acid rescued bile acid conjugation and reduced blood pressure in hypertensive rats. Subsequently, untargeted metabolomics uncovered altered energy metabolism following conjugation of bile acids as a mechanism alleviating high blood pressure.CONCLUSION:Together this work reveals conjugated bile acids as nutritionally re-programmable anti-hypertensive metabolites.

Supplementary Table S6. Univariate and multivariate Cox proportional hazard regression models of the 27-gene signature using the Kim (A), Lindgren (B), Sjodahl (C), and TCGA (D) data sets. Univariate Cox proportional hazard regression analysis showed that a gene signature of top 50% was significantly associated with increased risk of death in Kim, Lindgren, and Sjodahl cohorts (hazard ratio (HR) above 1). In a multivariate model, after adjusted for other factors such as sex, age and tumor stage showed that the 27 gene signature of top 50% was associated with increased risk of death in TCGA, Kim, and Lindgren. Tumor stage was significantly associated with survival in all TCGA, Kim, Sjodahl and Lindgren cohorts, and age was significantly associated with survival in TCGA and Kim cohorts. Hazard ratios (HRs) derived from Cox proportional hazards models are expressed with 95% confidence intervals (CIs) and p-values. For all analyses, p-value <0.05 was considered clinically significant and was highlighted in green.

Prostate cancer (PCa) is the second most common cancer and constitutes about 14.7% of total cancer cases. PCa is highly prevalent and more aggressive in African-American (AA) men than in European-American (EA) men. PCa tends to be highly heterogeneous, and its complex biology is not fully understood. We use metabolomics to better understand the mechanisms behind PCa progression and disparities in its clinical outcome. Adenosine deaminase (ADA) is a key enzyme in the purine metabolic pathway; it was found to be upregulated in PCa and is associated with higher-grade PCa and poor disease-free survival. The inosine-to-adenosine ratio, which is a surrogate for ADA activity was high in PCa patient urine and higher in AA PCa compared to EA PCa. To understand the significance of high ADA in PCa, we established ADA overexpression models and performed various in vitro and in vivo studies. Our studies have revealed that an acute increase in ADA expression during later stages of tumor development enhances in vivo growth in multiple pre-clinical models. Further analysis revealed that mTOR signaling activation could be associated with this tumor growth. Chronic ADA overexpression shows alterations in the cells' adhesion machinery and a decrease in cells' ability to adhere to the extracellular matrix in vitro. Losing cell-matrix interaction is critical for metastatic dissemination which suggests that ADA could potentially be involved in promoting metastasis. This is supported by the association of higher ADA expression with higher-grade tumors and poor patient survival. Overall, our findings suggest that increased ADA expression may promote PCa progression, specifically tumor growth and metastatic dissemination.

Supplementary Figure S2. Chromatographic reproducibility of metabolomics of liver samples over three technical replicates identified by LC-MS using HILIC positive, HILIC negative, and reverse phase positive ionization modes. B) Chromatographic reproducibility of lipidomics of liver samples over three technical replicates identified on the LC-MS using positive and, negative ionization modes.

Supplement Table S5. List of differential lipids and their classification.

Supplementary Table S1. List of internal standards for metabololomics and lipidomics, spiked across the various profiling platforms.

Supplementary Table S8. Patient characteristics of bladder specimens used for figure 4B, 4C, and 4G.

Supplementary Table S7. Univariate and multivariate Cox proportional hazard regression models of the TCGA (A), Lindgren (B), Sjodahl (C), and Kim (D), data sets using CPT1B gene expression. Univariate Cox proportional hazard regression analysis showed that CPT1B expression yields a Hazard ratios below 1 in TCGA, Lindgren, and Sjodahl, and is significantly associated with survival in TCGA cohort. The multivariate models after adjustment for other factors such as sex, age and tumor stage, revealed that low (HR less than 1) CPT1B expression was significantly associated with an increased risk of death in TCGA. Hazard ratios (HRs) derived from the Cox proportional hazards models are expressed with 95% confidence intervals (CIs) and p-values. For all analyses, p-value <0.05 was considered clinically significant and are highlighted in green.

Supplementary Table S2. List of gene-specific primers used for q-PCR.

Supplementary Figure S5. A) Survival analysis of the upregulated (11/27) gene signature in high grade BLCA from the Kim, Lindgren, Sjodahl, and TCGA coherts. B) Survival analysis of the upregulated (11/27) gene signature in low grade BLCA from the Kim, Lindgren, and Sjodahl coherts. Upregulated genes associated with poor survival in the TCGA high-grade (log-rank p<0.0041), the Lindgren high grade cohort (log-rank p<0.0298), and in the Sjodahl low-grade cohort (log-rank p<0.0418). (LG=low-grade, HG= high-grade).

Supplementary Figure S1. A) Quality control of 519 metabolites across three biological replicates of pooled liver sample extracts and run in hydrophilic interaction liquid chromatography (HILIC) positive, HILIC negative, and reverse phase positive ionization modes. B) Quality control (QC) of detected 19 lipids across three biological replicates of pooled sample extracts run in positive and negative ionization modes.

Supplementary Figure S6. Survival analysis of individual downregulated genes showed poor prognosis associated with CPT1B and PIGB in the Lindgren and Sjodahl cohorts; PLA2G4A in the Lindgren cohort; PIGV in the Kim and Sjodahl cohorts; ATP8B1 in the Kim and Sjodahl cohorts; INPP5D in the Kim and Sjodahl cohorts; PIGZ in the Kim and Sjodahl cohorts; PLCD3 in the Sjodahl cohort; and PLA2G10 in the Kim cohort.

Supplementary Figure S7. mRNA expression of CPT1B in the Kim (p<2.7Ã-10-4), Sjodahl (p<2.5Ã-10-7), and Lindgren (p<2.8Ã-10-13) cohorts.