Clinicopathologic Characteristics and Prognosis of ERBB2-Low Breast Cancer Among Patients in the National Cancer Database.

Importance: Given conflicting results regarding the prognosis of erb-b2 receptor tyrosine kinase 2 (ERBB2; formerly HER2 or HER2/neu)-low breast cancer, a large-scale, nationally applicable comparison of ERBB2-low vs ERBB2-negative breast cancer is needed. Objective: To investigate whether ERBB2-low breast cancer is a clinically distinct subtype in terms of epidemiological characteristics, prognosis, and response to neoadjuvant chemotherapy. Design/Participants/Setting: This retrospective cohort study was conducted using the National Cancer Database, including 1 136 016 patients in the US diagnosed with invasive breast cancer from January 1, 2010, to December 31, 2019, who had ERBB2-negative disease and had immunohistochemistry results available. ERBB2-low tumors were classified as having an immunohistochemistry score of 1+, or 2+ with a negative in situ hybridization test. Data were analyzed from November 1, 2021, through November 30, 2022. Exposures: Standard therapy according to routine clinical practice. Main Outcomes and Measures: The primary outcomes were overall survival (OS), reported as adjusted hazard ratios (aHRs), and pathologic complete response, reported as adjusted odds ratios (aORs), for ERBB2-negative vs ERBB2-low breast cancer, controlling for age, sex, race and ethnicity, Charlson-Deyo Comorbidity Index score, treatment facility type, tumor grade, tumor histology, hormone receptor status, and cancer stage. Results: The study identified 1 136 016 patients (mean [SD] age, 62.4 [13.1] years; 99.1% female; 78.6% non-Hispanic White), of whom 392 246 (34.5%) were diagnosed with ERBB2-negative and 743 770 (65.5%) with ERBB2-low breast cancer. The mean (SD) age of the ERBB2-negative group was 62.1 (13.2) years and 62.5 (13.0) years for the ERBB2-low group. Higher estrogen receptor expression was associated with increased rates of ERBB2-low disease (aOR, 1.15 per 10% increase). Compared with non-Hispanic White patients, of whom 66.1% were diagnosed with ERBB2-low breast cancer, fewer non-Hispanic Black (62.8%) and Hispanic (61.0%) patients had ERBB2-low disease, although in non-Hispanic Black patients this was mediated by differences in rates of triple-negative disease and other confounders. A slightly lower rate of pathologic complete response was seen in patients with ERBB2-low disease vs patients with ERBB2-negative disease on multivariable analysis (aOR, 0.89; 95% CI, 0.86-0.92; P < .001). ERBB2-low status was also associated with small improvements in OS for stage III (aHR, 0.92; 95% CI, 0.89-0.96; P < .001) and stage IV (aHR, 0.91; 95% CI, 0.87-0.96; P < .001) triple-negative breast cancer, although this amounted to only a 2.0% (stage III) and 0.4% (stage IV) increase in 5-year OS. Conclusions and Relevance: This large-scale retrospective cohort analysis found minimal prognostic differences between ERBB2-low and ERBB2-negative breast cancer. These findings suggest that, moving forward, outcomes in ERBB2-low breast cancer will be driven by ERBB2-directed antibody-drug conjugates, rather than intrinsic differences in biological characteristics associated with low-level ERBB2 expression. These findings do not support the classification of ERBB2-low breast cancer as a unique disease entity.

DAXX-inducing phytoestrogens inhibit ER+ tumor initiating cells and delay tumor development.

Recurrence of estrogen receptor (ER)-positive breast tumors despite curative-intent adjuvant therapy is thought to be due to enrichment of tumor initiating cells (TIC) during endocrine therapy (ET). Recently, it was identified that by antagonizing the ER, ET promotes rapid degradation of the death-associated factor 6 (DAXX) protein, which is necessary and sufficient to potently inhibit TICs. Thus, the goal of the current study was to identify a DAXX-inducing agent to inhibit TICs and prevent proliferation of the tumor. Phytoestrogens (naringenin, resveratrol, genistein, apigenin, and quercetin) were screened for DAXX protein expression, anti-TIC and anti-proliferative efficacy in vitro and in vivo. Specific DAXX-inducing phytoestrogens were tested to assess selectivity towards ERα and/or ERß. Results showed that phytoestrogens tested induced DAXX protein expression and inhibited survival of TICs from ER+ MCF-7 and T47D cells. Only naringenin, resveratrol, and quercetin did not stimulate total cell proliferation. Naringenin, resveratrol, but not quercetin inhibited survival of TICs in vitro and in vivo in a DAXX-dependent manner. Naringenin-induced DAXX protein expression and inhibition of TICs seemed to be more selective towards ERß while resveratrol was more selective through ERα. Naringenin or resveratrol inhibited the rate of tumor initiation and rate of tumor growth in a DAXX-dependent manner. These results suggest that a therapeutic approach using a phytoestrogen to induce DAXX protein expression could potently inhibit TICs within a tumor to delay or prevent tumor initiation. Therefore, a DAXX-promoting phytoestrogen should be explored for prevention of tumor progression in advanced disease and relapse in the adjuvant setting.

DAXX Suppresses Tumor-Initiating Cells in Estrogen Receptor-Positive Breast Cancer Following Endocrine Therapy.

Estrogen receptor (ER)-positive breast cancer recurrence is thought to be driven by tumor-initiating cells (TIC). TICs are enriched by endocrine therapy through NOTCH signaling. Side effects have limited clinical trial testing of NOTCH-targeted therapies. Death-associated factor 6 (DAXX) is a newly identified marker whose RNA expression inversely correlates with NOTCH in human ER+ breast tumor samples. In this study, knockdown and overexpression approaches were used to investigate the role of DAXX on stem/pluripotent gene expression, TIC survival in vitro, and TIC frequency in vivo, and the mechanism by which DAXX suppresses TICs in ER+ breast cancer. 17ß-Estradiol (E2)-mediated ER activation stabilized the DAXX protein, which was required for repressing stem/pluripotent genes (NOTCH4, SOX2, OCT4, NANOG, and ALDH1A1), and TICs in vitro and in vivo. Conversely, endocrine therapy promoted rapid protein depletion due to increased proteasome activity. DAXX was enriched at promoters of stem/pluripotent genes, which was lost with endocrine therapy. Ectopic expression of DAXX decreased stem/pluripotent gene transcripts to levels similar to E2 treatment. DAXX-mediated repression of stem/pluripotent genes and suppression of TICs was dependent on DNMT1. DAXX or DNMT1 was necessary to inhibit methylation of CpGs within the SOX2 promoter and moderately within the gene body of NOTCH4, NOTCH activation, and TIC survival. E2-mediated stabilization of DAXX was necessary and sufficient to repress stem/pluripotent genes by recruiting DNMT1 to methylate some promoters and suppress TICs. These findings suggest that a combination of endocrine therapy and DAXX-stabilizing agents may inhibit ER+ tumor recurrence. SIGNIFICANCE: Estradiol-mediated stabilization of DAXX is necessary and sufficient to repress genes associated with stemness, suggesting that the combination of endocrine therapy and DAXX-stabilizing agents may inhibit tumor recurrence in ER+ breast cancer.

Estrogen-mediated protection against coronary heart disease: The role of the Notch pathway.

Estrogen regulates a plethora of biological processes, under physiological and pathological conditions, by affecting key pathways involved in the regulation of cell proliferation, fate, survival and metabolism. The Notch receptors are mediators of communication between adjacent cells and are key determinants of cell fate during development and in postnatal life. Crosstalk between estrogen and the Notch pathway intervenes in many processes underlying the development and maintenance of the cardiovascular system. The identification of molecular mechanisms underlying the interaction between these types of endocrine and juxtacrine signaling are leading to a deeper understanding of physiological conditions regulated by these steroid hormones and, potentially, to novel therapeutic approaches to prevent pathologies linked to reduced levels of estrogen, such as coronary heart disease, and cardiotoxicity caused by hormone therapy for estrogen-receptor-positive breast cancer.

Inhibition of the development of N-nitrosomethylbenzylamine-induced esophageal tumors in rats by strawberries and aspirin, alone and in combination.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of two subtypes of esophageal cancer, with high incidence and mortality rates in developing countries. OBJECTIVE: The current study investigated the potential chemoprotective effects of strawberries and aspirin against the development of rat esophageal papillomas, the precursors to ESCC. METHODS: Using a prevention model, we administered study diets to rats before, during, and after N-nitrosomethylbenzylamine (NMBA) treatment. The effects of the four diets were evaluated: the control diet, 5% strawberry powder in the control diet, 0.01% aspirin in the drinking water, and the combination of strawberries and aspirin. At week 25, we euthanized all the rats and collected their esophagi to quantify tumor incidence, multiplicity, and burden, as well as for molecular analysis. RESULTS: Both strawberries and aspirin significantly decreased esophageal tumor multiplicity, with the combination causing the most robust suppression. Aspirin alone and the combination decreased the total tumor burden in the esophagus. None of the diets had a significant effect on tumor incidence or the expression of COX-1 and COX-2. Strawberries and aspirin, alone and in combination, significantly suppressed squamous epithelial cell proliferation (PCNA). CONCLUSIONS: Strawberries, aspirin, and their combination exhibit chemoprotective effects against NMBA-induced esophageal tumors in rats.

Inhibition of HER2 Increases JAGGED1-dependent Breast Cancer Stem Cells: Role for Membrane JAGGED1.

Purpose: HER2-positive breast cancer is driven by cells possessing stem-like properties of self-renewal and differentiation, referred to as cancer stem cells (CSC). CSCs are implicated in radiotherapy, chemotherapy resistance, and tumor recurrence. NOTCH promotes breast CSC survival and self-renewal, and overexpression of NOTCH1 and the NOTCH ligand JAGGED1 predict poor outcome. Resistance to anti-HER2 therapy in HER2+ breast cancer requires NOTCH1, and that combination of trastuzumab and a gamma secretase inhibitor (GSI) prevents tumor relapse in xenograft models.Experimental Design: The current study investigates mechanisms by which HER2 tyrosine kinase activity regulates NOTCH-dependent CSC survival and tumor initiation.Results: Lapatinib-mediated HER2 inhibition shifts the population of HER2+ breast cancer cells from low membrane JAGGED1 expression to higher levels, independent of sensitivity to anti-HER2 treatment within the bulk cell population. This increase in membrane JAGGED1 is associated with higher NOTCH receptor expression, activation, and enrichment of CSCs in vitro and in vivo Importantly, lapatinib treatment results in growth arrest and cell death of JAGGED1 low-expressing cells while the JAGGED1 high-expressing cells continue to cycle. High membrane JAGGED1 protein expression predicts poor overall cumulative survival in women with HER2+ breast cancer.Conclusions: These results indicate that higher membrane JAGGED1 expression may be used to either predict response to anti-HER2 therapy or for detection of NOTCH-sensitive CSCs posttherapy. Sequential blockade of HER2 followed by JAGGED1 or NOTCH could be more effective than simultaneous blockade to prevent drug resistance and tumor progression. Clin Cancer Res; 24(18); 4566-78. ©2018 AACR.

Notch-1-PTEN-ERK1/2 signaling axis promotes HER2+ breast cancer cell proliferation and stem cell survival.

Trastuzumab targets the HER2 receptor on breast cancer cells to attenuate HER2-driven tumor growth. However, resistance to trastuzumab-based therapy remains a major clinical problem for women with HER2+ breast cancer. Breast cancer stem cells (BCSCs) are suggested to be responsible for drug resistance and tumor recurrence. Notch signaling has been shown to promote BCSC survival and self-renewal. Trastuzumab-resistant cells have increased Notch-1 expression. Notch signaling drives cell proliferation in vitro and is required for tumor recurrence in vivo. We demonstrate herein a mechanism by which Notch-1 is required for trastuzumab resistance by repressing PTEN expression to contribute to activation of ERK1/2 signaling. Furthermore, Notch-1-mediated inhibition of PTEN is necessary for BCSC survival in vitro and in vivo. Inhibition of MEK1/2-ERK1/2 signaling in trastuzumab-resistant breast cancer cells mimics effects of Notch-1 knockdown on bulk cell proliferation and BCSC survival. These findings suggest that Notch-1 contributes to trastuzumab resistance by repressing PTEN and this may lead to hyperactivation of ERK1/2 signaling. Furthermore, high Notch-1 and low PTEN mRNA expression may predict poorer overall survival in women with breast cancer. Notch-1 protein expression predicts poorer survival in women with HER2+ breast cancer. These results support a potential future clinical trial combining anti-Notch-1 and anti-MEK/ERK therapy for trastuzumab-resistant breast cancer.

FOXO3, estrogen receptor alpha, and androgen receptor impact tumor growth rate and infiltration of dendritic cell subsets differentially between male and female mice.

Tumors evade immune recognition and destruction in many ways including the creation of an immune-suppressive tumor microenvironment (TME). Dendritic cells (DC) that infiltrate the TME are tolerogenic, and suppress effector T cells and anti-tumor activity. Previous reports demonstrated that a key regulator of tolerance in DC is the transcription factor FOXO3. Gender disparity has been studied in cancer in relation to incidence, aggressiveness, and prognosis. Few studies have touched on the importance in relation to impact on the immune system. In the current study, we show that there are significant differences in tumor growth between males and females. Additionally, frequencies and the function of FOXO3 expressed by DC subsets that infiltrate tumors vary between genders. Our results show for the first time that DC FOXO3 expression and function is altered in females. In vitro results indicate that these differences may be the result of exposure to estrogen. These differences may be critical considerations for the enhancement of immunotherapy for cancer.

Dietary Consumption of Black Raspberries or Their Anthocyanin Constituents Alters Innate Immune Cell Trafficking in Esophageal Cancer.

Freeze-dried black raspberries (BRB), their component anthocyanins (AC), and a metabolite of BRB ACs, protocatechuic acid (PCA), inhibit the development of esophageal cancer in rats induced by the carcinogen, N-nitrosomethylbenzylamine (NMBA). All three components reduce inflammation in the esophagus and in plasma. The present study determined the relation of changes in inflammatory markers to infiltration of innate immune cells into NMBA-treated esophagus. Rats were injected with NMBA (0.35 mg/kg) for 5 weeks while on control diet. Following NMBA treatment, rats were fed diets containing 6.1% BRB powder, an AC-rich fraction of BRBs (3.8 µmol/g), or 500 ppm PCA. At weeks 15, 25, and 35, inflammatory biomarker expression in the plasma and esophagus was quantified, and infiltration of immune cells in the esophagus was examined. At all three time points, BRB, AC, and PCA similarly affected cytokine production in the esophagus and plasma of NMBA-treated rats, relative to the NMBA-only control. These included decreased expression of the proinflammatory cytokine IL1ß and increased expression of the anti-inflammatory cytokine IL10. Moreover, all three diets also increased the expression of IL12, a cytokine that activates both cytolytic natural killer and CD8(+) T cells. In addition, the three diets also decreased infiltration of both macrophages and neutrophils into the esophagus. Overall, our results suggest that another mechanism by which BRBs, ACs, and PCA inhibit NMBA-induced esophageal tumorigenesis is by altering cytokine expression and innate immune cell trafficking into tumor tissues.

CCL22 to Activate Treg Migration and Suppress Depigmentation in Vitiligo.

In vitiligo, gradual cutaneous depigmentation and cytotoxic T-cell activity against melanocytes are accompanied by a paucity of regulatory T cells (Tregs) in vitiligo patient skin, indicating that autoimmune responses are not adequately held in check. Thus, we sought a means to repopulate patient skin with Tregs. We hypothesized that enhanced expression of CCL22 can promote Treg skin homing to suppress depigmentation. The mouse Ccl22 gene was cloned into an expression vector and resulting DNA was used for gene gun treatment. Two spontaneous depigmentation models with different kinetics of melanocyte loss were utilized, expressing tyrosinase-reactive and gp100-reactive TCR transgenes. Mice were subjected to five gene gun treatments 6 days apart, scanned for depigmentation weekly thereafter, and monitored for activation and proliferation of relevant T cells and for Treg infiltration to the skin. Significantly reduced depigmentation 2 weeks after treatment was accompanied by a markedly increased abundance of Tregs in the skin at the expense of melanocyte-reactive, TCR transgenic T cells, as well as by reduced proliferation and reduced IFN-γ production in response to cognate peptide. Continued treatment may be necessary for sustained, local immunosuppression. These findings suggest that topical CCL22 may be used for the treatment of vitiligo.

Chemoprevention of esophageal cancer with black raspberries, their component anthocyanins, and a major anthocyanin metabolite, protocatechuic acid.

Diets containing either freeze-dried black raspberries (BRBs) or their polyphenolic anthocyanins (ACs) have been shown to inhibit the development of N-nitrosomethylbenzylamine (NMBA)-induced esophageal cancer in rats. The present study was conducted to determine whether PCA, a major microbial metabolite of black raspberry (BRB) ACs, also prevents NMBA-induced esophageal cancer in rats. F344 rats were injected with NMBA three times a week for 5 weeks and then fed control or experimental diets containing 6.1% BRBs, an anthocyanin (AC)-enriched fraction derived from BRBs, or protocatechuic acid (PCA). Animals were exsanguinated at weeks 15, 25, and 35 to quantify the development of preneoplastic lesions and tumors in the esophagus, and to relate this to the expression of inflammatory biomarkers. At weeks 15 and 25, all experimental diets were equally effective in reducing NMBA-induced esophageal tumorigenesis, as well as in reducing the expression of pentraxin-3 (PTX3), a cytokine produced by peripheral blood mononuclear cells in response to interleukin (IL)-1ß and TNF-α. All experimental diets were also active at reducing tumorigenesis at week 35; however, the BRB diet was significantly more effective than the AC and PCA diets. Furthermore, all experimental diets inhibited inflammation in the esophagus via reducing biomarker (COX-2, iNOS, p-NF-κB, and sEH) and cytokine (PTX3) expression. Overall, our data suggest that BRBs, their component ACs, and PCA inhibit NMBA-induced esophageal tumorigenesis, at least in part, by their inhibitory effects on genes associated with inflammation.