Monika L. Burness

Epidermal Growth Factor Receptor in Triple-Negative and Basal-Like Breast Cancer: Promising Clinical Target or Only a Marker?

Triple-negative breast cancers represent a subset of breast cancers with a particularly aggressive phenotype and poor clinical outcomes. Recent molecular profiling of these tumors has revealed a high frequency of epidermal growth factor receptor (EGFR) dysregulation, among other abnormalities. EGFR status correlates negatively with survival in patients with triple-negative breast cancers, and thus focus has turned on this receptor as a potential clinical target. Two classes of EGFR inhibitors are currently in clinical use: the monoclonal antibodies and the small molecule tyrosine kinase inhibitors. Trials of these drugs in breast cancer, however, have been largely disappointing. It remains to be seen whether advances in our understanding of the mechanisms of EGFR dysregulation and effects of multiple compensatory pathways in breast cancer, coupled with improved targeting to appropriate patient populations, will yield meaningful improvements in clinical outcomes.

The stem cell niche in health and malignancy

Somatic stem cells play a well-defined and important role in tissue renewal. Their malignant counterparts, cancer stem cells, are thought to be responsible for tumor initiation and possibly chemotherapy resistance, although controversy remains regarding both the origin and characterization of these cells. Both somatic and cancer stem cells appear to occupy specialized microenvironments in many organs. These niches are important for both maintenance of quiescence and control of cellular survival and proliferation. Targeting cancer stem cells and their microenvironments may provide new therapies to eradicate tumors. The efficacy of several drugs in current use is mediated at least in part via effects on the microenvironment, and new drugs that target the niche are currently in clinical trials.

Tunable Thermal-Sensitive Polymer–Graphene Oxide Composite for Efficient Capture and Release of Viable Circulating Tumor Cells

A highly sensitive microfluidic system to capture circulating tumor cells from whole blood of cancer patients is presented. The device incorporates graphene oxide into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. By decreasing the temperature, captured cells may be released for subsequent analysis.

Dormant breast cancer micrometastases reside in specific bone marrow niches that regulate their transit to and from bone

Breast cancer cells traffic to and from the peripheral blood within specific vascular niches, and this migration can be therapeutically targeted. Taking away cancer’s hideouts Breast cancer is notorious for its ability to relapse after many years, long after a patient had completed treatment. Price et al. demonstrate that the culprits responsible for such late metastasis may be dormant cancer cells hiding in perivascular niches. The authors showed that proteins called E-selectin and CXCR4 exert different forces on these cancer cells, with CXCR4 anchoring breast cancer cells to their niches and E-selectin allowing entry of cancer cells into the bone marrow. These findings suggest that combining a CXCR4 inhibitor to force the cells out of their niches and an E-selectin inhibitor to prevent metastasis to the bone marrow could help trap the cells in the vasculature, where they could be killed with chemotherapy. Breast cancer metastatic relapse can occur years after therapy, indicating that disseminated breast cancer cells (BCCs) have a prolonged dormant phase before becoming proliferative. A major site of disease dissemination and relapse is bone, although the critical signals that allow circulating BCCs to identify bone microvasculature, enter tissue, and tether to the microenvironment are poorly understood. Using real-time in vivo microscopy of bone marrow (BM) in a breast cancer xenograft model, we show that dormant and proliferating BCCs occupy distinct areas, with dormant BCCs predominantly found in E-selectin– and stromal cell–derived factor 1 (SDF-1)–rich perisinusoidal vascular regions. We use highly specific inhibitors of E-selectin and C-X-C chemokine receptor type 4 (CXCR4) (SDF-1 receptor) to demonstrate that E-selectin and SDF-1 orchestrate opposing roles in BCC trafficking. Whereas E-selectin interactions are critical for allowing BCC entry into the BM, the SDF-1/CXCR4 interaction anchors BCCs to the microenvironment, and its inhibition induces mobilization of dormant micrometastases into circulation. Homing studies with primary BCCs also demonstrate that E-selectin regulates their entry into bone through the sinusoidal niche, and immunohistochemical staining of patient BMs shows dormant micrometastatic disease adjacent to SDF-1+ vasculature. These findings shed light on how BCCs traffic within the host, and suggest that simultaneous blockade of CXCR4 and E-selectin in patients could molecularly excise dormant micrometastases from the protective BM environment, preventing their emergence as relapsed disease.

RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer.

Introduction: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)–deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-negative breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display “cancer stem cell” properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. Experimental Design: We determined the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. Results: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1–wild-type cells were relatively resistant to PARP inhibition alone, but reduction of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. Conclusions: Our data suggest that in both BRCA1-mutant and BRCA1–wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514–22. ©2016 AACR.

Triple negative breast cancer in BRCA1 mutation carriers with a complete radiologic response to neoadjuvant paclitaxel: a case report.

While good therapies exist for treating breast cancer in the adjuvant setting, many patients still experience disease recurrence, and improved data regarding ways to tailor therapy to individual patients is needed. When treating BRCA-associated breast cancers, it has been suggested that BRCA-associated cancers have predicable responses to various chemotherapeutic agents. We present two cases in which BRCA-1 mutation carriers had complete responses to paclitaxel chemotherapy, a drug that is predicted to have poor efficacy in this patient population. These cases underscore the need for more research to further understand the complex biology of these tumors, and for larger clinical trials to test potential therapeutic options.

Paclitaxel Plasma Concentration after the First Infusion Predicts Treatment-Limiting Peripheral Neuropathy

Purpose: Paclitaxel exposure, specifically the maximum concentration (Cmax) and amount of time the concentration remains above 0.05 μmol/L (Tc>0.05), has been associated with the occurrence of paclitaxel-induced peripheral neuropathy. The objective of this study was to validate the relationship between paclitaxel exposure and peripheral neuropathy. Experimental Design: Patients with breast cancer receiving paclitaxel 80 mg/m2 × 12 weekly doses were enrolled in an observational clinical study (NCT02338115). Paclitaxel plasma concentration was measured at the end of and 16–26 hours after the first infusion to estimate Cmax and Tc>0.05. Patient-reported peripheral neuropathy was collected via CIPN20 at each dose, and an 8-item sensory subscale (CIPN8) was used in the primary analysis to test for an association with Tc>0.05. Secondary analyses were conducted using Cmax as an alternative exposure parameter and testing each parameter with a secondary endpoint of the occurrence of peripheral neuropathy–induced treatment disruption. Results: In 60 subjects included in the analysis, the increase in CIPN8 during treatment was associated with baseline CIPN8, cumulative dose, and relative dose intensity (P < 0.05), but neither Tc>0.05 (P = 0.27) nor Cmax (P = 0.99). In analyses of the secondary endpoint, cumulative dose (OR = 1.46; 95% confidence interval (CI), 1.18–1.80; P = 0.0008) and Tc>0.05 (OR = 1.79; 95% CI, 1.06–3.01; P = 0.029) or Cmax (OR = 2.74; 95% CI, 1.45–5.20; P = 0.002) were associated with peripheral neuropathy–induced treatment disruption. Conclusions: Paclitaxel exposure is predictive of the occurrence of treatment-limiting peripheral neuropathy in patients receiving weekly paclitaxel for breast cancer. Studies are warranted to determine whether exposure-guided dosing enhances treatment effectiveness and/or prevents peripheral neuropathy in these patients. Clin Cancer Res; 24(15); 3602–10. ©2018 AACR.

Abstract 4226: Histone deacetylase inhibitors sensitize cancer stem cells to PARP inhibitors in triple-negative breast cancer

Background: Triple negative breast cancers (TNBCs) have a poor prognosis and are a therapeutic challenge due to resistance to multiple chemotherapy drugs. Growing evidence suggests that cancer stem cells (CSCs) may be promising therapeutic targets for treating TNBCs. Poly ADP ribose polymerase (PARP) inhibitors have shown striking activity in preclinical models of BRCA-deficient breast carcinomas. However they are less efficacious in tumors without germline BRCA mutations, which account for more than 80% of all TNBCs. Our data demonstrates that treatment of TNBC with PARP inhibitor increases CSCs, likely due to an elevated homologous recombination (HR) repair mechanism. Several small studies have suggested that histone deacetylase (HDAC) inhibitors, which decrease HR repair proteins, may sensitize non-BRCA-mutated TNBC to PARP inhibitors. Currently, little is known whether this synergistic effect can reverse the increase in CSCs caused by PARP inhibition. In this study, our aim was to investigate if HDAC inhibitors could sensitize CSCs to PARP inhibitors in TNBCs. Results: Using four TNBC cell lines, we confirmed the synthetic lethality of HDAC and PARP inhibitors (Vorinostat and Olaparib respectively) in vitro. CompuSyn analysis revealed Combination Indices of 0.66, 0.44, 0.47 and 0.16 in SUM159, MDAMB231, SUM149 and HCC1937 cells respectively at ED50. An ALDEFLUOR assay revealed ∼1.9 fold increase in the CSC population after PARP inhibition in BRCA-mutant SUM149 and HCC1937 cells, while the absolute CSC number, (total cell number times percentage of ALDEFLUOR-positive cells) remained unchanged. Whereas in BRCA-wild type TNBC cell lines SUM159 and MDAMB231, there was no change in CSCs. Addition of the HDAC inhibitor decreased absolute CSC number by 85%, 78% and 40% in SUM149, SUM159 and HCC1937 cells respectively after 7 days of PARP inhibition. Although the mechanism is not fully understood at present, we demonstrated that Rad51, a key player in HR repair, mediates the sensitivity of TNBCs to PARP inhibition, and HDAC inhibitor prevents the formation of Rad51 foci at DNA damage site. This may indicate that HDAC inhibition sensitizes the CSCs of TNBCs to PARP inhibition via suppressing the HR pathway. Conclusion: Our data suggests that PARP inhibition only targets the bulk cancer cells in BRCA-mutant TNBC cells while sparing CSCs and has no effect on BRCA WT cells. HDAC inhibition sensitized CSCs of TNBC to PARP inhibition regardless of BRCA status, possibly via interfering with Rad51 function. The inhibitors we used in the present study are either FDA-approved or at Phase III clinical trials, and the combination is predicted to have low toxicity. Thus, our findings are promising and could be rapidly translated into clinical use. Citation Format: Yajing Liu, Rachel Martin-Trevino, Li Shang, April Davis, Max Wicha, Suling Liu, Monika Burness. Histone deacetylase inhibitors sensitize cancer stem cells to PARP inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4226. doi:10.1158/1538-7445.AM2015-4226

Tamoxifen and ERα36: Fertilizing the seeds of breast cancer metastasis

Hormonal therapies including tamoxifen have been a mainstay for the treatment of breast tumors that express the classical estrogen receptor ERα66. Wang et al. now report that tamoxifen may paradoxically stimulate ALDH-expressing breast cancer stem cells via activation of the ERα66 variant ERα36. One of the greatest advances in breast cancer therapy has been the stratification of tumors into subtypes based on expression of estrogen receptor (ER), progesterone receptor (PR), and HER2. Tumors that express ER, known as ER-positive breast cancers, are the most common subtype of breast cancer, accounting for ~ 70% of early-stage breast cancer diagnoses. They are highly curable, with 20-year disease-free survival rates approaching 80% for earliest stage tumors. Estrogen signaling is mediated by binding of 17β-estradiol (E2) to ER, which initiates a signaling cascade resulting in downstream activation of numerous transcription factors. Two estrogen receptors, ERα and ERβ, have been described to signal via genomic and non-genomic pathways. These two receptors regulate unique genes, and in fact can serve opposing functions. Current clinically-utilized ER assays test for the primary alpha receptor, ERα66. Several other ERs and related proteins have been described. For example, estrogen related receptor beta (ERRβ) is found in embryonic stem cells, and is involved in cellular selfrenewal. ERα36, a truncated variant of ERα66 without transcriptional activity, has been described and is thought to function via a non-genomic signaling pathway. Interestingly, the expression of ERα36 is independent of ERα66, and thus it is expressed in a subset of cells in both ER-positive and ER-negative breast cancers. The mainstay of therapy for ER-positive tumors is endocrine therapy, consisting of tamoxifen or aromatase inhibitors. Endocrine therapies work via preventing estrogen stimulation of the estrogen receptor and downstream signaling, although by different mechanisms. Aromatase inhibitors inhibit peripheral conversion of androgens into estrogen via the aromatase enzyme, thereby decreasing circulating levels of estradiol. Tamoxifen, a selective estrogen receptor modulator (SERM), acts as an estrogen antagonist in certain tissues, including breast tissue. While endocrine therapy is highly effective, some tumors recur in spite of endocrine therapy. Cancer stem cells (CSCs) are postulated as a mechanism of tumor recurrence. These cells, which comprise a small portion of most ER-positive tumors, are thought to mediate tumor initiation, metastasis, and treatment resistance. Furthermore, the important clinical issue of dormancy in ER breast cancer has been attributed to this cell population. In ER-positive tumors, CSCs identified by the expression of aldehyde dehydrogenase (ALDH1) are often found to be negative for ERα66, and the mitogenic effects of estrogen on this cell population have been thought to be indirect involving paracrine effects. In a recent paper in Cell Research, Wang et al. add to the evolving story the importance of ERα36 in breast cancer with a report that stimulation of ERα36 promotes metastasis via promotion of ALDH1A1-positive CSCs. 4 This group has previously reported that expression of ERα36 is related to resistance to tamoxifen, and that estrogen signaling via ERα36 regulates the maintenance of breast CSCs. In the current study, they analyzed ERα36 expression in 1677 breast cancer samples, and convincingly showed that ERα36 expression was correlated with tumor size, grade, and lymph node involvement. They further found that ERα36-positive tumors were more likely to recur as metastatic disease, regardless of ERα66 status. In patients who received tamoxifen, those with ERα36-positive tumors showed shorter metastasis-free survival than those with ERα36-negative tumors, although this may in fact be due to ERα36 itself, not a negative effect of tamoxifen therapy. In contrast to the effects of tamoxifen, they show that the clinical benefit of aromatase inhibitors is independent of ERα36 expression. They conclude that these differential clinical outcomes are due to the stimulatory effect of tamoxifen on ERα36-expressing cells, a finding consistent with but not proven by the clinical data. They present compelling pre-clinical data linking ERα36 and CSCs and demonstrate that tamoxifen stimulates ERα36expressing CSCs, increasing their capacity for migration, invasion, and tumor initiation. Furthermore, they show that this occurs via the regulation of ALDH expression. It has previously been demonstrated that ALDH is a marker of breast CSC and a predictor of poor clinical outcome. The current work adds increasing evidence for an important functional role of ALDH. It also suggests that estrogen may have a direct mitogenic effect on breast CSC which is mediated by ERα36. In addition, this study suggests that tamoxifen resistance may in part be mediated by ERα36, and that CSCs are responsible for the effect. The authors propose that tumors could be screened for ERα36 expression, and that tamoxifen should not be used for tumors that express ERα36. However, this suggestion is counter to numerous clinical studies demonstrating the effectiveness of tamoxifen in both therapeutic and preventative settings. A meta-analysis published by the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) in ER-positive breast cancer demonstrated ~ 30% reduction in breast cancer mortality with 5 years of tamoxifen therapy.

Abstract 3307: Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients

Introduction and Objective: The metastasis of cancer is preceded by the dissemination of cancer cells from the primary tumor site to remote sites via the blood circulation. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival. Immune-affinity based capture, although being the most commonly used method for the isolation of CTCs, offers low throughput (∼1mL/hr) and have considerably cell loss caused by the heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing the physical properties of CTCs have been developed to overcome the limitations, such as micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yielding the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis. Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of CTCs is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 5 mL of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques. Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples. Conclusion: The study of CTCs could have a direct impact upon society by presenting novel ways to address one of the major hurdles in cancer research - early detection - and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics. Citation Format: Eric Lin, Lianette Rivera, Shamileh Fouladdel, Hyeun Joong Yoon, Stephanie Guthrie, Jacob Weiner, Yadwinder S. Deol, Evan Keller, Vaibhav Sahai, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3307.