Supplemental Figure 3 Racial difference in survival in (A) Luminal A compared to (B) TNBC breast cancer. CI, confidence interval.

Supplemental Figure 5 Overall breast cancer survival based on median income of county of diagnosis in European American (EA) (A) compared to African American (AA) patients (B). CI, confidence interval.

The use of digital pathology for the histomorphologic profiling of pathological specimens is expanding the precision and specificity of quantitative tissue analysis at an unprecedented scale; thus, enabling the discovery of new and functionally relevant histological features of both predictive and prognostic significance. In this study, we apply quantitative automated image processing and computational methods to profile the subcellular distribution of the multi-functional transcriptional regulator, Kaiso ( ZBTB33 ), in the tumors of a large racially diverse breast cancer cohort from a designated health disparities region in the United States. Multiplex multivariate analysis of the association of Kaiso’s subcellular distribution with other breast cancer biomarkers reveals novel functional and predictive linkages between Kaiso and the autophagy-related proteins, LC3A/B, that are associated with features of the tumor immune microenvironment, survival, and race. These findings identify effective modalities of Kaiso biomarker assessment and uncover unanticipated insights into Kaiso’s role in breast cancer progression.

553 Background: gp78, also known as the autocrine motility factor receptor (AMFR) or RNF45, is a polytopic RING-type E3 ubiquitin ligase resident to the endoplasmic reticulum (ER) that plays major role in the cellular response to stress by regulating ER homeostasis and signaling through its participation in the unfolded protein response (UPR) and ER associated degradation. We used machine learning (ML) and statistical modeling (SM) to assess gp78 as a protein biomarker that is an independent predictor of breast cancer (bc) survival exclusively in women of self-reported African descent as opposed to European ancestry. Methods: We examined a cohort of racially diverse 555 BC bc patients who underwent surgery for their primary BC in Greenville, NC using ML and SM approach. We leveraged the availability of RNA-seq gene expression data on a portion of our bc cohort (N=136 of 555) to construct gene expression signatures. Results: Using antibodies developed in the Weissman lab and established methods for quantitative IHC, we have found that gp78 expression is significantly increased in the tumors of bc patients compared to normal breast epithelia. In addition, we found that gp78 is expressed at significantly higher levels in bc of non-Hispanic black women (NHB) compared to non-Hispanic white women (NHW) (p=0.0038), and that bc subtypes known to be more aggressive and associated with higher grades like, Basal (p=1.6e-12), Luminal B (p=2.3e-4) and HER2(8.3e-4), display significantly higher levels of gp78 compare to Luminal A. Moreover, Kaplan-Meier survival curve analyses show that gp78 protein expression is more significantly associated with poor survival in NHB women (HR:1.65, p=0.073) compared to NHW women (HR:2.01, p=0.004). Finally, multivariate analysis reveals that gp78 protein expression, based on quantitative IHC, is an independent predictor of poor bc survival exclusively in women of African (NHB) ancestry (HR:1.99, p=0.017). We leveraged the availability of RNA-seq gene expression data on a portion of our bc cohort to construct gene expression signatures or gene modules. An analysis of pooled publicly available data from 845 patients that underwent neoadjuvant chemotherapy for bc (primarily taxane and anthracycline based), reveals that gp78 gene modules are highly predictive of patient response to therapy. gp78-derived gene modules show both high fold difference and significance in predicting response to therapy (AUC:0.72) which is very similar to other multi-gene panels that are currently in clinical use including Prosigna, MammaPrint, and Oncotype Dx. Conclusions: Our results show that gp78/AMFR is an independent predictor of bc survival and response to therapy, based on race, thus implicating a role for this protein, and potentially the UPR, as underlying biological differences in tumor properties linked to genetic ancestry.

The disparity in breast cancer burden of African Americans compared with European Americans is one of the most revealing instances in oncology associated with ethnicity. Recent studies demonstrated that there were highly intratumoral genetic heterogeneity and increased the frequency of basal subtypes among African Americans than European Americans. Because significant race-based disparities in breast cancer patients with hormone-receptor positive still persistent even after reflecting socio-economic status, there may be substantial roles playing by intrinsic biological factors contributing to the disparities. Transcriptional regulation governed by estrogen receptor plays critical roles in major changes in chromatin landscape that facilitate the assembly of other transcriptional complexes affecting mammary tumor initiation and proliferation. We investigated the estrogen receptor and its essential pioneer transcription factors how their expressions are associated with racial disparity with their prognostic significance. Co-expression analysis of estrogen receptor and its pioneer transcription factors suggest that ER-positive patients of women of European ancestry show much more favorable survival than women of African ancestry. Differential comparison of protein expression integrated with network-level gene expression analysis reveals significant differences in the predictive ability of the luminal master regulators based on race and survival. Moreover, we identified genes in the downstream of these master regulators that are highly correlated with race and survival. The comparative analysis of the predictive value of race-based cutoffs suggests that these master regulators of luminal differentiation have reduced transcriptional activity in the downstream regulatory network pathways in African ancestry. Functional characterization of the luminal master regulators and their downstream pathways may provide a better understanding of the intrinsic mechanisms that drive racial disparities in breast cancer survival. Citation Format: Jung S. Byun, Sandeep Singhal, Samson Park, Dae IK Yi, Ambar Caban, Nasreen Vohra, Eliseo J. Perez-Stable, Anna Napoles, Kevin L. Gardner. Transcription regulatory networks associated with luminal master regulator expression and breast cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5220.

The burden of cancer in the United States is unevenly spread across its different populations, with stark differences in both disease prevalence and outcome on the basis of race and ethnicity. Although a large portion of these differences can be explained by a variety of sociobehavioral and socioeconomic factors, even after these exposures are taken into consideration, considerable disparities persist. In this review, we explore a conceptual framework of biological theories and unifying concepts, based on an evolutionary perspective, that may help better define common guiding principles for exploration of under Lying causes of cancer health disparities. The ultimate goal of this conceptual perspective is to outline approaches that may aid in establishing integrated pathway and processes analyses to provide useful insights to guide the development of future interventions. These interventions will improve outcome, increase prevention, and ultimately eliminate all disparities.

CtBP (Carboxyl‐terminal binding protein) is a dimeric nuclear protein and an epigenetic regulator that is activated in the presence of NADH to recruit and target a variety of histone modifying complexes and transcriptional regulators to chromatin, thus providing a defined mechanism through which carbohydrate metabolism can drive epigenetic regulatory events. We have recently shown that CtBP drives epithelial‐mesenchymal transition (EMT), genome instability, and acquisition of tumor‐initiating stem cell‐like pathways in breast cancer. Recently, we observed that elevated CtBP protein expression is correlated with poor survival of breast cancer patients in two independent cohorts. This finding led us to study further what complexes are associated with CtBP in breast cancer patient samples and whether such complexes could be linked to pathways that influence clinical outcomes. Immunoprecipitation assay of CtBP followed by mass spectrometry revealed CtBP's interactions with BRG1 (SMARCA4) and ZNF217 in the MCF‐7 breast cancer cell line. ZNF217 is a Zinc Finger DNA binding Transcription factor located on chromosome 20q13. ZNF217, amplified in 8–30% of breast cancers, is capable of transforming primary mammary epithelial cells, has been seen in hyperplastic mammary lesions, and plays a major role in TGF‐beta signaling pathways. BRG1 is a subunit of the SWI/SNF chromatin remodeling complex. The SWI/SNF is an ATP‐dependent chromatin remodeling complex that plays a role in a variety of cancers. Most notably, its subunits are mutated in 20% of cancers, with lesions that either disrupt tumor suppressor activity or confer oncogenic properties on the SWI/SNF complex. To assess whether these complexes form in patient tumor samples, we employed the Proximity Ligation Assay (PLA) technology. PLA enables detection of protein:protein interactions with sensitivity at the level of single molecule resolution in tissue. We performed PLA on formalin fixed and paraffin embedded (FFPE) tumor samples from breast cancer patients and could demonstrate significant levels of CtBP1:BRG1, CtBP2:BRG1 and ZNF217:CtBP1 interactions. Recent studies from our lab and those of others suggest a cooperative interaction between CtBP and BRG1 in the induction of drug resistance genes. To assess functional relationship between CtBP and BRG1 in patient samples, we measured the transcript abundance of the multiple drug resistance gene MDR1 (ABCB1) in patient samples stratified by BRG1 and CtBP expression. The findings show a significant correlation between MDR1 transcript abundance in patients with both high levels of BRG1 and CtBP (P‐value = 0.021). This data suggest future applications of BRG1/CtBP PLA as a possible screen for the activation of drug resistance pathways in breast cancer patients. Finally, we provide evidence that adopting this assay to tissue microarray (TMA) format is highly feasible.

In the United States, more than 70% of adults are either overweight or obese. This epidemic is associated with grave health consequences, including cardiovascular diseases, kidney diseases, and cancer. One possible mechanism accounting for these risks is metabolic imbalance associated with obesity. Currently, the most common method of quantifying metabolic imbalance is by measuring a person's body mass index (BMI), an effective but incomplete measurement that only takes into account the overall height and weight. An approach that may complement the quantification of obesity‐induced metabolic imbalance is directly examining adipocyte size distribution around the tumors. After adolescence, the adipose cell number remains relatively steady, and the excess energy storage is primarily accomplished through adipocyte hypertrophy; however, after prolonged obesity and continuous positive energy balance, the adipocytes begin to undergo hyperplasia to increase their number. Therefore, we believe the state of obesity with a high level of adipocyte hyperplasia may represent the worst prognostic case of metabolic imbalance. Because 70% of human breast is composed of adipose tissue, this is especially relevant for breast cancer. Furthermore, a key molecule that may work in conjunction with obesity to worsen breast cancer prognosis is C‐terminal binding protein (CtBP), a dimeric nuclear protein that plays a substantial epigenetic role in modifying gene expression by inducing Epithelial‐Mesenchymal Transition, genome instability, and stem cell‐like pathways. What makes CtBP metabolically relevant is that its activity depends on NADH, a prominent byproduct of metabolism that is elevated under conditions of obesity, diabetes, and metabolic syndrome. When NADH binds to CtBP, CtBP is able to dimerize, improve its stability, and enhance its nuclear localization. For this reason, we think CtBP may be a key metabolic sensor that connects modifiable risk factors like obesity to molecular pathways of breast cancer.In this study, we explore a link between obesity and CtBP by examining cohorts of breast cancer patient samples. Using digital analysis of histological sections and immunohistochemistry, we first measure adipocyte cross‐sectional area and obtain CtBP protein expression for each patient. We analyze and correlate the adipocyte size distribution and BMI with gene and protein expression of CtBP. Our preliminary results demonstrate that obesity measured by BMI exacerbates a poor breast cancer patient outcome driven by high protein level of CtBP alone. Our data also suggest that breast cancer patients with high BMI and excessive hyperplasia have significantly poorer survival and higher protein expression of CtBP. The results of this study provide new approaches to understanding how aspects of lifestyle and personal traits may interact with the molecular pathways of CtBP to influence breast cancer incidence and mortality.

The C-terminal binding protein (CtBP) is an NADH-dependent dimeric family of nuclear proteins that scaffold interactions between transcriptional regulators and chromatin-modifying complexes. Its association with poor survival in several cancers implicates CtBP as a promising target for pharmacological intervention. We employed computer-assisted drug design to search for CtBP inhibitors, using quantitative structure-activity relationship (QSAR) modeling and docking. Functional screening of these drugs identified 4 compounds with low toxicity and high water solubility. Micro molar concentrations of these CtBP inhibitors produces significant de-repression of epigenetically silenced pro-epithelial genes, preferentially in the triple-negative breast cancer cell line MDA-MB-231. This epigenetic reprogramming occurs through eviction of CtBP from gene promoters; disrupted recruitment of chromatin-modifying protein complexes containing LSD1, and HDAC1; and re-wiring of activating histone marks at targeted genes. In functional assays, CtBP inhibition disrupts CtBP dimerization, decreases cell migration, abolishes cellular invasion, and improves DNA repair. Combinatorial use of CtBP inhibitors with the LSD1 inhibitor pargyline has synergistic influence. Finally, integrated correlation of gene expression in breast cancer patients with nuclear levels of CtBP1 and LSD1, reveals new potential therapeutic vulnerabilities. These findings implicate a broad role for this class of compounds in strategies for epigenetically targeted therapeutic intervention.

Abstract The C-terminal binding protein (CtBP) is a family of dimeric nuclear proteins whose levels are increased in cancers of the colon, ovaries, prostate and breast. Elevated CtBP expression is associated with poor cancer survival and can also distinguish those node negative breast cancer patients who will show worse survival. This implicates CtBP as both a biomarker and a promising candidate for therapeutic intervention. As a dimer, CtBP provides a scaffold that couples multiple different DNA-binding transcriptional regulators with a variety of chromatin modifying protein complexes to alter the epigenetic landscape throughout the nucleus. These properties provide the rationale for pharmacological targeting of CtBP to change epigenetically regulated gene expression in cancer cells. In this study, we employ computer assisted drug design to screen for optimal quantitative structure-activity relationships (QSARs) between small molecules and CtBP to identify 24 potential CtBP inhibitors. Functional screening of these compounds identifies 4 lead compounds with low toxicity and high water solubility. Treatment of breast cancer cells at micro-molar concentrations of these small molecular inhibitors induces significant de-repression of epigenetically silenced pro-epithelial genes in the mesenchymal, triple negative breast cancer cell line, MDA-MB-231. This re-activation is associated with eviction of CtBP from the respective gene promoters, disrupted recruitment of CtBP-chromatin modifying protein complexes, increased deposition of activating epigenetic histone marks, and upregulation of both pro-epithelial gene mRNA and protein expression. In functional assays, CtBP inhibition by these small molecular inhibitors decreases cellular invasion, and improves DNA repair. FRET (Förster resonance energy transfer) analysis demonstrates that CTBP inhibition results in decreased FRET intensity, suggesting that CTBP dimerization is repressed by CTBP inhibition. In addition, pharmacological inhibition of CtBP combines with established epigenetically targeted drugs to synergistically decrease cell migration and potentiate the reactivation of silenced pro-epithelial gene expression in triple negative cancer cells. Finally, CTBP inhibition results in transcriptional repression of MDR1 expression and reduces the population of Doxorubicin resistant cells in the triple negative breast cancer cell lines. These findings implicate the possible use of this class of compounds in strategies for therapeutic intervention that may increase the efficacy and decrease the acquired resistance to targeted therapeutic intervention in breast cancer. Citation Format: Jung S. Byun, Samson Park, Dae IK Yi, Mohamed Kabbout, Genqing Liang, Kevin L. Gardner. Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1156. doi:10.1158/1538-7445.AM2017-1156