Mammary stem cell and macrophage markers are enriched in normal tissue adjacent to inflammatory breast cancer.

INTRODUCTION: We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. MATERIALS AND METHODS: Normal breast parenchyma ≥ 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44+CD49f+CD133/2+ mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. RESULTS: 8 of 8 IBC samples expressed isolated CD44+CD49f+CD133/2+ stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44+CD49f+CD133/2+ cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). CONCLUSIONS: Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

Epidemiological risk factors associated with inflammatory breast cancer subtypes.

BACKGROUND: In this single-institution case-control study, we identified risk factors associated with inflammatory breast cancer (IBC) subtypes based on staining of estrogen receptor (ER), progesterone receptor (PR) and expression of human epidermal growth factor 2 (HER2neu) to determine distinct etiologic pathways. METHODS: We identified 224 women with IBC and 396 cancer-free women seen at the MD Anderson Cancer Center. Multinomial logistic regression was used to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) for associations between breast cancer risk factors and the IBC tumor subtypes: luminal (ER+ and/or PR+/HER2neu-), HER2neu+ (any ER and PR, HER2neu+), and triple-negative (ER-/PR-/HER2neu-). RESULTS: In multivariable analysis, compared with women age ≥26 at first pregnancy, women age <26 had a higher risk of triple-negative IBC (OR 3.32, 95% CI 1.37-8.05). Women with a history of breast-feeding had a lower risk of triple-negative (OR 0.30; 95% CI 0.15-0.62) and luminal IBC (OR 0.35, 95% CI 0.18-0.68). A history of smoking was associated with an increased risk of luminal IBC (OR 2.37; 95% CI 1.24-4.52). Compared with normal-weight women, those who were overweight or obese (body mass index ≥25 kg/m(2)) had a higher risk of all three tumor subtypes (p < 0.01 for all subtypes). CONCLUSION: Overweight or obese status is important modifiable risk factor for IBC of any subtype. Modifiable risk factors, age at first pregnancy (≥26), breast-feeding, and smoking may be associated with specific IBC subtypes. These results highlight the importance of evaluating epidemiologic risk factors for IBC for the identification of subtype-specific prevention strategies.

Cancer stem cell markers are enriched in normal tissue adjacent to triple negative breast cancer and inversely correlated with DNA repair deficiency.

INTRODUCTION: We hypothesized that cells present in normal tissue that bear cancer stem cell markers may represent a cancer cell of origin or a microenvironment primed for tumor development, and that their presence may correlate with the clinically defined subtypes of breast cancer that show increased tumorigenicity and stem cell features. methods: Normal tissues sampled at least 5 cm from primary tumors (normal adjacent tissue) were obtained from 61 chemotherapy-naive patients with breast cancer treated with mastectomy. Samples were stained simultaneously with immunofluorescence for CD44/CD49f/CD133/2 stem cell markers. We assessed the association between CD44+CD49f+CD133/2+ staining in normal adjacent tissue and breast cancer receptor subtype (defined by the expression of the estrogen (ER), progesterone (PR), or human epidermal growth factor-2 (Her2) receptors). We also examined the correlation between CD44+CD49f+CD133/2+ immunofluorescence and each of two previously published gene signatures, one derived from stem-cell enriched tissue and one from BRCA mutated tissue expected to have defective DNA repair. RESULTS: Patients with triple negative breast cancer (ER­/PR­/HER2­) expressed CD44+CD49f+CD133/2+ in 9 of 9 normal adjacent tissue samples compared with 7 of 52 ER+ and/or Her2+ tumors (P < 0.001). Further, expression of CD44+CD49f+CD133/2+ by normal adjacent tissue correlated positively with a stem cell-derived tumorigenic signature (P <0.001) and inversely with a defective DNA-repair signature (P <0.001). CONCLUSION: Normal cells bearing cancer stem cell markers are associated with the triple negative receptor subtype of breast cancer. This study suggests stem cell staining and gene expression signatures from normal breast tissues represent novel tissue-based risk biomarkers for triple negative breast cancer. Validation of these results in additional studies of normal tissue from cancer-free women could lay the foundation for future targeted triple negative breast cancer prevention strategies.

Selective targeting of radiation-resistant tumor-initiating cells.

Tumor-initiating cells (TICs) have been shown both experimentally and clinically to be resistant to radiation and chemotherapy, potentially resulting in residual disease that can lead to recurrence. In this study, we demonstrate that TICs isolated from p53 null mouse mammary tumors repair DNA damage following in vivo ionizing radiation more efficiently than the bulk of the tumor cells. Down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was observed both in fluorescence activated cell sorting (FACS)-isolated TICs as compared to non-TICs and in TIC-enriched mammospheres as compared to primary tumor cells depleted of TICs. This effect was accompanied by increased Akt signaling, as well as by the direct activation of the canonical Wnt/beta-catenin signaling pathway specifically within the TIC subpopulation by phosphorylation of beta-catenin on serine 552. Using limiting dilution transplantation performed on p53 null tumor cells transduced with Wnt reporter lentivirus, we demonstrated that FACS sorting of cells expressing TOP-eGFP resulted in a marked enrichment for TICs. Furthermore, FACS analysis demonstrated that cells with active Wnt signaling overlapped with the TIC subpopulation characterized previously using cell surface markers. Finally, pharmacological inhibition of the Akt pathway in both mammospheres and syngeneic mice bearing tumors was shown to inhibit canonical Wnt signaling as well as the repair of DNA damage selectively in TICs, sensitizing them to ionizing radiation treatment. Thus, these results suggest that pretreatment with Akt inhibitors before ionizing radiation treatment may be of potential therapeutic benefit to patients.

Improving Daytime Functioning, Work Performance, and Quality of Life in Postmenopausal Women With Insomnia: Comparing Cognitive Behavioral Therapy for Insomnia, Sleep Restriction Therapy, and Sleep Hygiene Education.

STUDY OBJECTIVES: Insomnia is a chief complaint among postmenopausal women, and insomnia impairs daytime functioning and reduces quality of life. Recent evidence supports the efficacy of cognitive behavioral therapy for insomnia (CBTI) for menopausal insomnia, but it remains unclear whether treating insomnia improves daytime function in this population. This study evaluated whether CBTI improves daytime fatigue, energy, self-reported sleepiness, work productivity, and quality of life in postmenopausal women with insomnia, and whether sleep restriction therapy (SRT)-a single component of CBTI-is equally efficacious. METHODS: Single-site, randomized control trial. One hundred fifty postmenopausal women (56.44 ± 5.64 years) with perimenopausal or postmenopausal onset or exacerbation of chronic insomnia were randomized to 3 treatment conditions: sleep hygiene education control (SHE), SRT, and CBTI. Blinded assessments were performed at pretreatment, posttreatment, and 6-month follow-up. RESULTS: CBTI and SRT produced moderate-to-large improvements in fatigue, energy, sleepiness, and work function at posttreatment and 6 months later. The CBTI group reported better quality of life as indicated by substantial improvements in emotional wellbeing and resiliency to physical and emotional problems, whereas the SRT and SHE groups only showed improvements in resiliency to physical problems. Pain complaints decreased as sleep improved but were not associated with specific treatment conditions. Similarly, insomnia remitters reported fewer daytime and nighttime hot flashes, although reductions were not associated with any specific treatment. CONCLUSIONS: CBTI and SRT are efficacious options for postmenopausal women with chronic insomnia. Both interventions improve daytime function, quality of life, and work performance, although CBTI produces superior results including the added benefit of improved emotional health. CLINICAL TRIAL REGISTRATION: Registry: ClinicalTrials.gov; Title: Behavioral Treatment of Menopausal Insomnia; Sleep and Daytime Outcomes; Identifier: NCT01933295; URL: https://clinicaltrials.gov/ct2/show/record/NCT01933295.

High-density and very-low-density lipoprotein have opposing roles in regulating tumor-initiating cells and sensitivity to radiation in inflammatory breast cancer.

PURPOSE: We previously demonstrated that cholesterol-lowering agents regulate radiation sensitivity of inflammatory breast cancer (IBC) cell lines in vitro and are associated with less radiation resistance among IBC patients who undergo postmastectomy radiation. We hypothesized that decreasing IBC cellular cholesterol induced by treatment with lipoproteins would increase radiation sensitivity. Here, we examined the impact of specific transporters of cholesterol (ie lipoproteins) on the responses of IBC cells to self-renewal and to radiation in vitro and on clinical outcomes in IBC patients. METHODS AND MATERIALS: Two patient-derived IBC cell lines, SUM 149 and KPL4, were incubated with low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL), or high-density lipoproteins (HDL) for 24 hours prior to irradiation (0-6 Gy) and mammosphere formation assay. Cholesterol panels were examined in a cohort of patients with primary IBC diagnosed between 1995 and 2011 at MD Anderson Cancer Center. Lipoprotein levels were then correlated to patient outcome, using the log rank statistical model, and examined in multivariate analysis using Cox regression. RESULTS: VLDL increased and HDL decreased mammosphere formation compared to untreated SUM 149 and KPL4 cells. Survival curves showed enhancement of survival in both of the IBC cell lines when pretreated with VLDL and, conversely, radiation sensitization in all cell lines when pretreated with HDL. In IBC patients, higher VLDL values (>30 mg/dL) predicted a lower 5-year overall survival rate than normal values (hazard ratio [HR] = 1.9 [95% confidence interval [CI]: 1.05-3.45], P=.035). Lower-than-normal patient HDL values (<60 mg/dL) predicted a lower 5-year overall survival rate than values higher than 60 mg/dL (HR = 3.21 [95% CI: 1.25-8.27], P=.015). CONCLUSIONS: This study discovered a relationship among the plasma levels of lipoproteins, overall patient response, and radiation resistance in IBC patients and IBC patient-derived cell lines. A more expansive study is needed to verify these observations.

EZH2 expression correlates with locoregional recurrence after radiation in inflammatory breast cancer.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2), a member of the polycomb group proteins, has been shown to promote cancer progression and breast cancer stem cell (CSC) expansion. Breast CSCs are associated with resistance to radiation in inflammatory breast cancer (IBC), a rare but aggressive variant of breast cancer. In this retrospective study, we examined the clinical role of EZH2 in locoregional recurrence (LRR) of IBC patients treated with radiation. PATIENTS AND METHODS: 62 IBC patients who received radiation (7 pre-operative, 55 post-operative) and had adequate follow up to assess LRR were the subject of this study. Positive EZH2 status was defined as nuclear immunohistochemical staining in at least 10% of invasive cancer cells. Association of EZH2 expression with clinicopathologic features were evaluated using the Chi-square statistic and actuarial LRR free survival (LRFS) was determined using the Kaplan-Meier method. RESULTS: The median follow-up for this cohort was 33.7 months, and the 5-year overall LRFS rate was 69%. Of the 62 patients, 16 (25.8%) had LRR, and 15 out of 16 LRR occurred in EZH2 expressing cases. Univariate analysis indicated that patients who had EZH2-positive IBC had a significantly lower 5-year locoregional free survival (LRFS) rate than patients who had EZH2-negative IBC (93.3% vs. 59.1%; P = 0.01). Positive EZH2 expression was associated significantly with negative ER status (97.1% in ER- vs 48.1% in ER+; P < 0.0001) and triple-negative receptor status (P = 0.0001) and all triple-negative tumors were EZH2-positive. In multivariate analysis, only triple negative status remained an independent predictor of worse LRFS (hazard ratio 5.64, 95% CI 2.19 - 14.49, P < 0.0001). CONCLUSIONS: EZH2 correlates with locoregional recurrence in IBC patients who received radiation treatment. EZH2 expression status may be used in addition to receptor status to identify a subset of patients with IBC who recur locally in spite of radiation and may benefit from enrollment in clinical trials testing radiosensitizers.

Separation by cell size enriches for mammary stem cell repopulation activity.

Mammary gland reconstitution experiments, as well as lineage tracing experiments, have provided evidence for the existence of adult mammary stem cells (MaSCs). In addition, cell sorting techniques for specific cell surface markers (CD24(+)CD29(H)CD49f(H)Sca1(-)) have been used to prospectively isolate MaSC-enriched populations. Although these markers enrich for cell subpopulations that harbor MaSCs, they do not identify regenerative stem cells uniquely. Here, we report that MaSCs can be further defined by the property of cell size. Fluorescence-activated cell sorting was used to analyze sizing beads and further separate populations of cells with varying degrees of forward scatter (FSC). Cells with a low FSC that were approximately <10 µm in size lacked outgrowth potential and failed to reconstitute the mammary gland when transplanted into the cleared fat pads of syngeneic mice. In contrast, cells >10 µm in size with a higher FSC had increased outgrowth potential as compared with lineage-negative (LIN(-)) control cells. Limiting dilution transplantation assays indicated that the repopulating ability of LIN(-)CD24(+)CD29(H) cells that were >10 µm in size was significantly increased as compared with cells marked by CD24 and CD29 alone. These results suggest that MaSCs can be further isolated by sorting based on size/FSC. These findings have critical implications for understanding mammary gland stem cell biology, an important requisite step for understanding the etiology of breast cancer.

The antihelmintic drug pyrvinium pamoate targets aggressive breast cancer.

WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.

Thermal enhancement with optically activated gold nanoshells sensitizes breast cancer stem cells to radiation therapy.

Breast cancer metastasis and disease recurrence are hypothesized to result from residual cancer stem cells, also referred to as tumor-initiating cells, which evade initial treatment. Using both syngeneic mouse and human xenograft models of triple-negative breast cancer, we have demonstrated that a subpopulation enriched in cancer stem cells was more resistant to treatment with 6 gray of ionizing radiation than the bulk of the tumor cells, and accordingly their relative proportion increased 48 to 72 hours after ionizing radiation treatment. In contrast, we achieved a larger reduction in tumor size without a concomitant increase in the percentage of cancer stem cells by treating with local hyperthermia for 20 minutes at 42°C after ionizing radiation using intravenously administered, optically activated gold nanoshells. Forty-eight hours after treatment, cells derived from the tumors treated with ionizing radiation plus hyperthermia exhibited both a marked decrease in tumorigenicity and a more differentiated phenotype than mock- and ionizing radiation-treated tumors. Thus, we have confirmed that these cancer stem cells are responsible for accelerated repopulation in vivo and demonstrated that hyperthermia sensitizes this cell population to radiation treatment. These findings suggest that local hyperthermia delivered by gold nanoshells plus radiation can eliminate radioresistant breast cancer stem cells.

Identification of tumor-initiating cells in a p53-null mouse model of breast cancer.

Using a syngeneic p53-null mouse mammary gland tumor model that closely mimics human breast cancer, we have identified, by limiting dilution transplantation and in vitro mammosphere assay, a Lin(-)CD29(H)CD24(H) subpopulation of tumor-initiating cells. Upon subsequent transplantation, this subpopulation generated heterogeneous tumors that displayed properties similar to the primary tumor. Analysis of biomarkers suggests the Lin(-)CD29(H)CD24(H) subpopulation may have arisen from a bipotent mammary progenitor. Differentially expressed genes in the Lin(-)CD29(H)CD24(H) mouse mammary gland tumor-initiating cell population include those involved in DNA damage response and repair, as well as genes involved in epigenetic regulation previously shown to be critical for stem cell self-renewal. These studies provide in vitro and in vivo data that support the cancer stem cell (CSC) hypothesis. Furthermore, this p53-null mouse mammary tumor model may allow us to identify new CSC markers and to test the functional importance of these markers.