Racial differences in breast cancer outcomes by hepatocyte growth factor pathway expression.

PURPOSE: Black women have a 40% increased risk of breast cancer-related mortality. These outcome disparities may reflect differences in tumor pathways and a lack of targetable therapies for specific subtypes that are more common in Black women. Hepatocyte growth factor (HGF) is a targetable pathway that promotes breast cancer tumorigenesis, is associated with basal-like breast cancer, and is differentially expressed by race. This study assessed whether a 38-gene HGF expression signature is associated with recurrence and survival in Black and non-Black women. METHODS: Study participants included 1957 invasive breast cancer cases from the Carolina Breast Cancer Study. The HGF signature was evaluated in association with recurrence (n = 1251, 171 recurrences), overall, and breast cancer-specific mortality (n = 706, 190/328 breast cancer/overall deaths) using Cox proportional hazard models. RESULTS: Women with HGF-positive tumors had higher recurrence rates [HR 1.88, 95% CI (1.19, 2.98)], breast cancer-specific mortality [HR 1.90, 95% CI (1.26, 2.85)], and overall mortality [HR 1.69; 95% CI (1.17, 2.43)]. Among Black women, HGF positivity was significantly associated with higher 5-year rate of recurrence [HR 1.73; 95% CI (1.01, 2.99)], but this association was not significant in non-Black women [HR 1.68; 95% CI (0.72, 3.90)]. Among Black women, HGF-positive tumors had elevated breast cancer-specific mortality [HR 1.80, 95% CI (1.05, 3.09)], which was not significant in non-Black women [HR 1.52; 95% CI (0.78, 2.99)]. CONCLUSION: This multi-gene HGF signature is a poor-prognosis feature for breast cancer and may identify patients who could benefit from HGF-targeted treatments, an unmet need for Black and triple-negative patients.

Hepatocyte growth factor pathway expression in breast cancer by race and subtype.

BACKGROUND: African American women have the highest risk of breast cancer mortality compared to other racial groups. Differences in tumor characteristics have been implicated as a possible cause; however, the tumor microenvironment may also contribute to this disparity in mortality. Hepatocyte growth factor (HGF) is a stroma-derived marker of the tumor microenvironment that may affect tumor progression differentially by race. OBJECTIVE: To examine whether an HGF gene expression signature is differentially expressed by race and tumor characteristics. METHODS: Invasive breast tumors from 1957 patients were assessed for a 38-gene RNA-based HGF gene expression signature. Participants were black (n = 1033) and non-black (n = 924) women from the population-based Carolina Breast Cancer Study (1993-2013). Generalized linear models were used to estimate the relative frequency differences (RFD) in HGF status by race, clinical, and demographic factors. RESULTS: Thirty-two percent of tumors were positive for the HGF signature. Black women were more likely [42% vs. 21%; RFD = + 19.93% (95% CI 16.00, 23.87)] to have HGF-positive tumors compared to non-black women. Triple-negative patients had a higher frequency of HGF positivity [82% vs. 13% in non-triple-negative; RFD = + 65.85% (95% CI 61.71, 69.98)], and HGF positivity was a defining feature of basal-like subtype [92% vs. 8% in non-basal; RFD = + 81.84% (95% CI 78.84, 84.83)]. HGF positivity was associated with younger age, stage, higher grade, and high genomic risk of recurrence (ROR-PT) score. CONCLUSION: HGF expression is a defining feature of basal-like tumors, and its association with black race and young women suggests it may be a candidate pathway for understanding breast cancer disparities.

A functional role for the cancer disparity-linked genes, CRYßB2 and CRYßB2P1, in the promotion of breast cancer.

BACKGROUND: In the USA, the breast cancer mortality rate is 41% higher for African-American women than non-Hispanic White women. While numerous gene expression studies have classified biological features that vary by race and may contribute to poorer outcomes, few studies have experimentally tested these associations. CRYßB2 gene expression has drawn particular interest because of its association with overall survival and African-American ethnicity in multiple cancers. Several reports indicate that overexpression of the CRYßB2 pseudogene, CRYßB2P1, and not CRYßB2 is linked with race and poor outcome. It remains unclear whether either or both genes are linked to breast cancer outcomes. This study investigates CRYßB2 and CRYßB2P1 expression in human breast cancers and breast cancer cell line models, with the goal of elucidating the mechanistic contribution of CRYßB2 and CRYßB2P1 to racial disparities. METHODS: Custom scripts for CRYßB2 or CRYßB2P1 were generated and used to identify reads that uniquely aligned to either gene. Gene expression according to race and tumor subtype were assessed using all available TCGA breast cancer RNA sequencing alignment samples (n = 1221). In addition, triple-negative breast cancer models engineered to have each gene overexpressed or knocked out were developed and evaluated by in vitro, biochemical, and in vivo assays to identify biological functions. RESULTS: We provide evidence that CRYßB2P1 is expressed at higher levels in breast tumors compared to CRYßB2, but only CRYßB2P1 is significantly increased in African-American tumors relative to White American tumors. We show that independent of CRYßB2, CRYßB2P1 enhances tumorigenesis in vivo via promoting cell proliferation. Our data also reveal that CRYßB2P1 may function as a non-coding RNA to regulate CRYßB2 expression. A key observation is that the combined overexpression of both genes was found to suppress cell growth. CRYßB2 overexpression in triple-negative breast cancers increases invasive cellular behaviors, tumor growth, IL6 production, immune cell chemoattraction, and the expression of metastasis-associated genes. These data underscore that both CRYßB2 and CRYßB2P1 promote tumor growth, but their mechanisms for tumor promotion are likely distinct. CONCLUSIONS: Our findings provide novel data emphasizing the need to distinguish and study the biological effects of both CRYßB2 and CRYßB2P1 as both genes independently promote tumor progression. Our data demonstrate novel molecular mechanisms of two understudied, disparity-linked molecules.

Silver nanoparticles alter epithelial basement membrane integrity, cell adhesion molecule expression, and TGF-ß1 secretion.

Silver nanoparticles (AgNPs) are widely used in consumer and pharmaceutical products due to their antipathogenic properties. However, safety concerns have been raised due to their bioactive properties. While reports have demonstrated AgNPs can embed within the extracellular matrix, their effects on basement membrane (BM) production, integrin engagement, and tissue-integrity are not well-defined. This study analyzed the effects of AgNPs on BM production, composition and integrin/focal adhesion interactions in representative lung, esophageal, breast and colorectal epithelia models. A multidisciplinary approach including focused proteomics, QPCR arrays, pathway analyses, and immune-based, structural and functional assays was used to identify molecular and physiological changes in cell adhesions and the BM induced by acute and chronic AgNP exposure. Dysregulated targets included CD44 and transforming growth factor-beta, two proteins frequently altered during pathogenesis. Results indicate AgNP exposure interferes with BM and cell adhesion dynamics, and provide insight into the mechanisms of AgNP-induced disruption of epithelial physiology.

Race-associated biological differences among luminal A and basal-like breast cancers in the Carolina Breast Cancer Study.

BACKGROUND: We examined racial differences in the expression of eight genes and their associations with risk of recurrence among 478 white and 495 black women who participated in the Carolina Breast Cancer Study Phase 3. METHODS: Breast tumor samples were analyzed for PAM50 subtype and for eight genes previously found to be differentially expressed by race and associated with breast cancer survival: ACOX2, MUC1, FAM177A1, GSTT2, PSPH, PSPHL, SQLE, and TYMS. The expression of these genes according to race was assessed using linear regression and each gene was evaluated in association with recurrence using Cox regression. RESULTS: Compared to white women, black women had lower expression of MUC1, a suspected good prognosis gene, and higher expression of GSTT2, PSPHL, SQLE, and TYMS, suspected poor prognosis genes, after adjustment for age and PAM50 subtype. High expression (greater than median versus less than or equal to median) of FAM177A1 and PSPH was associated with a 63% increase (hazard ratio (HR) = 1.63, 95% confidence interval (CI) = 1.09-2.46) and 76% increase (HR = 1.76, 95% CI = 1.15-2.68), respectively, in risk of recurrence after adjustment for age, race, PAM50 subtype, and ROR-PT score. Log2-transformed SQLE expression was associated with a 20% increase (HR = 1.20, 95% CI = 1.03-1.41) in recurrence risk after adjustment. A continuous multi-gene score comprised of eight genes was also associated with increased risk of recurrence among all women (HR = 1.11, 95% CI = 1.04-1.19) and among white (HR = 1.14, 95% CI = 1.03-1.27) and black (HR = 1.11, 95% CI = 1.02-1.20) women. CONCLUSIONS: Racial differences in gene expression may contribute to the survival disparity observed between black and white women diagnosed with breast cancer.

Race-associated biological differences among Luminal A breast tumors.

African-American (AA) women have higher breast cancer-specific mortality rates. A higher prevalence of the worse outcome Basal-like breast cancer subtype contributes to this, but AA women also have higher mortality even within the more favorable outcome Luminal A breast cancers. These differences may reflect treatment or health care access issues, inherent biological differences, or both. To identify potential biological differences by race among Luminal A breast cancers, gene expression data from 108 CAU and 57 AA breast tumors were analyzed. Race-associated genes were evaluated for associations with survival. Finally, expression of race- and survival-associated genes was evaluated in normal tissue of AA and CAU women. Six genes (ACOX2, MUC1, CRYBB2, PSPH, SQLE, TYMS) were differentially expressed by race among Luminal A breast cancers and were associated with survival (HR <0.8, HR >1.25). For all six genes, tumors in AA had higher expression of poor prognosis genes (CRYBB2, PSPH, SQLE, TYMS) and lower expression of good prognosis genes (ACOX2, MUC1). A score based on all six genes predicted survival in a large independent dataset (HR = 1.9 top vs. bottom quartile, 95% CI: 1.4-2.5). For four genes, normal tissue of AA and CAU women showed similar expression (ACOX2, MUC1, SQLE, TYMS); however, the poor outcome-associated genes CRYBB2 and PSPH were more highly expressed in AA versus CAU women's normal tissue. This analysis identified gene expression differences that may contribute to mortality disparities and suggests that among Luminal A breast tumors there are biological differences between AA and CAU patients. Some of these differences (CRYBB2 and PSPH) may exist from the earliest stages of tumor development, or may even precede malignancy.

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer.

BACKGROUND: Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. METHODS: In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. RESULTS: Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. CONCLUSIONS: Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer.

Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ.

INTRODUCTION: Basal-like and luminal breast cancers have distinct stromal-epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal-epithelial interactions evolve in benign and pre-invasive lesions. METHODS: To study epithelial-stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis. RESULTS: Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basallike subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival. CONCLUSIONS: Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.

Linear and nonlinear rheology of liberase-treated breast cancer tumors.

Extracellular matrix (ECM) rigidity has been shown to increase the invasive properties of breast cancer cells, promoting transformation and metastasis through mechanotransduction. Reducing ECM stiffness via enzymatic digestion could be a promising approach to slowing breast cancer development by de-differentiation of breast cancer cells to less aggressive phenotypes and enhancing the effectiveness of existing chemotherapeutics via improved drug penetrance throughout the tumor. In this study, we examine the effects of injectable liberase (a blend of collagenase and thermolysin enzymes) treatments on the linear and nonlinear rheology of allograft 4T1 mouse mammary tumors. We perform two sets of in vivo mouse studies, in which either one or multiple treatment injections occur before the tumors are harvested for rheological analysis. The treatment groups in each study consist of a buffer control, free liberase enzyme in buffer, a thermoresponsive copolymer called LiquoGel (LQG) in buffer, and a combined, localized injection of LQG and liberase. All tumor samples exhibit gel-like linear rheological behavior with the elastic modulus significantly larger than the viscous modulus and both independent of frequency. Tumors that receive a single injection of localized liberase have significantly lower tumor volumes and lower tissue moduli at both the center and edge compared to buffer- and free liberase-injected control tumors, while tissue viscoelasticity remains relatively unaffected. Tumors injected multiple times with LQG and liberase also have lower tissue volumes but possess higher tissue moduli and lower viscoelasticities compared to the other treatment groups. We propose that a mechanotransductive mechanism could cause the formation of smaller but stiffer tumors after repeated, localized liberase injections. Large amplitude oscillatory shear (LAOS) experiments are also performed on tissues from the multiple injection study and the results are analyzed using MITlaos. LAOS analysis reveals that all 4T1 tumors from the multiple injection study exhibit nonlinear rheological behavior at high strains and strain rates. Examination of the Lissajous-Bowditch curves, Chebyshev coefficient ratios, elastic moduli, and dynamic viscosities demonstrate that the onset and type of nonlinear behavior is independent of treatment type and elastic modulus, suggesting that multiple liberase injections do not affect the nonlinear viscoelasticity of 4T1 tumors.

Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo.

INTRODUCTION: Macrophages comprise an essential component of the mammary microenvironment necessary for normal gland development. However, there is no viable in vivo model to study their role in normal human breast function. We hypothesized that adding primary human macrophages to the murine mammary gland would enhance and provide a novel approach to examine immune-stromal cell interactions during the humanization process. METHODS: Primary human macrophages, in the presence or absence of ectopic estrogen stimulation, were used to humanize mouse mammary glands. Mechanisms of enhanced humanization were identified by cytokine/chemokine ELISAs, zymography, western analysis, invasion and proliferation assays; results were confirmed with immunohistological analysis. RESULTS: The combined treatment of macrophages and estrogen stimulation significantly enhanced the percentage of the total gland humanized and the engraftment/outgrowth success rate. Timecourse analysis revealed the disappearance of the human macrophages by two weeks post-injection, suggesting that the improved overall growth and invasiveness of the fibroblasts provided a larger stromal bed for epithelial cell proliferation and structure formation. Confirming their promotion of fibroblasts humanization, estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion in vitro, as well as significantly increased proliferating cell nuclear antigen (PCNA) positive cells in humanized glands. Cytokine/chemokine ELISAs, zymography and western analyses identified TNFα and MMP9 as potential mechanisms by which estrogen-stimulated macrophages enhanced humanization. Specific inhibitors to TNFα and MMP9 validated the effects of these molecules on fibroblast behavior in vitro, as well as by immunohistochemical analysis of humanized glands for human-specific MMP9 expression. Lastly, glands humanized with macrophages had enhanced engraftment and tumor growth compared to glands humanized with fibroblasts alone. CONCLUSIONS: Herein, we demonstrate intricate immune and stromal cell paracrine interactions in a humanized in vivo model system. We confirmed our in vivo results with in vitro analyses, highlighting the value of this model to interchangeably substantiate in vitro and in vivo results. It is critical to understand the signaling networks that drive paracrine cell interactions, for tumor cells exploit these signaling mechanisms to support their growth and invasive properties. This report presents a dynamic in vivo model to study primary human immune/fibroblast/epithelial interactions and to advance our knowledge of the stromal-derived signals that promote tumorigenesis.