Metastatic Human Breast Cancer Research Articles

Abstract LB-035: 3D microfluidic tumor model for investigating metastatic tumor migration and colonization of secondary sites

Abstract Introduction: The ability to model the microenvironment of primary and secondary tumor sites is critical for advancing treatment options for metastatic breast cancer. There are currently no 3D tumor models that mimic the in vivo vascular geometry and tumor microenvironment for monitoring extravasation, tissue invasion, and colonization of secondary sites by metastatic tumors. In this study, we report on the development of a 3D tumor model with primary and secondary tumor sites, where metastatic breast cancer cells can escape the primary site, circulate in circulation, adhere to the vascular endothelium and subsequently invade distant tissues. Materials and Methods: Multiple synthetic tumor networks comprising primary and secondary tumor sites were developed using in vivo images and fabricated using soft lithography. Fluorescent dyes (FITC-dextran) was used to test the fluidic integrity while nano and micro-particles were used to test the porosity of the developed devices. Primary vascular endothelial cells were cultured in the vascular channels while GFP-labeled metastatic human breast cancer cell line (MDA-MB-231/GFP) or non-metastatic (MCF-7/GFP) was cultured in the primary tumor site in a 3D environment using Matrigel™. The vascular networks were perfused with endothelial cell media under physiological fluid flow conditions. Real-time monitoring of cellular growth, extravasation and invasion was performed using fluorescence microscopy over a four week period. Results and Discussion: Metastatic MDA-MB-231 breast cancer cells proliferated rapidly in the primary tumor site in contrast to non-metastatic MCF-7. Tumor cells were found to initiate a metastatic cascade as early as 7-10 days following growth in the primary site comprising of intravasation into the vascular channels, rolling and adhesion on the endothelium and extravasation into the secondary site. Significant differences were observed on the tumor behavior based on the flow profiles and morphology of the vascular networks. These results provide a unique perspective of the in vivo realism in an in vitro system for monitoring metastasis Conclusions: We have developed a 3D vascularized model for monitoring the invasive growth and the metastatic potential of tumors thereby mimicking the in vivo microenvironment of solid tumors. This model can be used to investigate tumor-endothelial cell interactions using a combination of real-time imaging techniques and screening of targeted therapeutics that may reduce the metastatic potential of tumors. Citation Format: Deborah Ramsey, Dustin Haithcock, Charles Garson, Ashley Gilbert, Ketan Bhatt, Balabhaskar Prabhakarpandian, Kapil Pant. 3D microfluidic tumor model for investigating metastatic tumor migration and colonization of secondary sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-035.

Abstract 3726: Design of personalised polymer based combination therapeutics for advanced stage breast cancer patients

Abstract Our objective is to engineer tumour-targeted polymer-based combination therapies specifically designed to treat metastatic breast cancer (BC) in a personalised manner. Our strategy is to develop novel multicomponent polymer conjugates and assess structure activity relationships in clinically relevant models to understand mechanisms of action. Furthermore, we are searching for novel drug combinations, including tumour-derived exosome release pathway inhibitors due to their association with metastasis and tumour drug resistance mechanisms. NCA polymerization techniques have allowed us to precisely control the synthesis of well-defined star-based (STP) and linear (LTP) polypeptidic architectures. After fluorescence labelling, we studied our systems in vitro demonstrating a much more rapid uptake for STP through clathrin-coated mediated endocytosis. pK and biodistribution in healthy mice revealed renal excretion profiles and greater terminal and accumulation half-lives for STP architectures when compared to the LTP. Remarkably, we also observed clear accumulation in immune system-related organs including the spleen and lymph nodes (LN) (up to 40% ID/g tissue accumulation in the LN after i.v. administration). This is possibly due to their inherent structural and morphological features, such as their size (≈100nm), highly negative z-potential values, and/or hydrophilic surfaces. These data highlight the great potential of our stabilized self-assemblies as carriers to target LN metastasis, cancer immunotherapy, or immune system-related approaches, such as vaccination. In parallel, we have performed a High Throughput Screening to select synergistic drug combinations to be used in polymer-based combination approaches through rationally designed linkers that confer adequate drug release kinetics. To perform this approach we selected four metastatic human BC cell lines representing clinical BC subtypes. All cell models have been fully characterized regarding their Cathepsin B activity, intracellular pH, as well as oestrogen, progesterone, Her2 receptors, GSH and exosomes levels; all representing patient-specific biomarkers. Cell viability and exosomes release modulation have been studied following treatments and several drug combinations have been selected for each specific BC subtype. With selected drug combinations different linking chemistry has been designed. We have studied a combination conjugate with the chemotherapeutic agent conjugated to PGA through two different length pH-labile hydrazone linkers. This provided different therapeutic outputs in cells and in a metastatic immunocompetent orthotopic breast cancer model, not only for the primary tumor but also for metastasis progression. The results obtained so far open up a wide range of opportunities for the currently unsuccessful clinical approaches to target LN metastasis and cancer immunotherapy. Citation Format: Ana Armiñán, Zoraida Andreu, Juan J Arroyo-Crespo, David Charbonnier, Esther Masiá, Fernanda Rodriguez-Otomin, Aroa Duro-Castano, Vicent J Nebot, Maria J Vicent. Design of personalised polymer based combination therapeutics for advanced stage breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3726.

Celecoxib inhibits mitochondrial O2 consumption, promoting ROS dependent death of murine and human metastatic cancer cells via the apoptotic signalling pathway

Capecitabine induced toxicities such as hand-foot syndrome (HFS) and progression of metastatic cancer are both treatable with concurrent celecoxib as shown in the ADAPT (Activating Cancer Stem Cells from Dormancy And Potentiate for Targeting) trial. In the present study, five commonly used NSAIDs, including celecoxib were compared for their pro-oxidative capacities as cytotoxic drugs against human and mouse metastatic melanoma or breast cancer cells in vitroand the source of cellular ROS production induced by celecoxib was examined in greater detail. ResultsCelecoxib was unique among the NSAIDs in that it showed particular potency as a cytotoxic drug against the metastatic cancer cells with IC50 values in the low micromolar range. Celecoxib rapidly enhanced mitochondrial superoxide production in situ from cancer cells within minutes, leading to a decrease in cellular respiration and dissipation of the mitochondrial transmembrane potential (Δψm), followed by extensive ROS-dependent apoptosis of the metastatic cancer cells. Celecoxib also showed rapid and direct effects on isolated mitochondria, inducing extensive ROS production in a dose-dependent manner, whilst it inhibited respiration via Complex I or Complex II when tested in whole cells. Mitochondrial ROS production was necessary for the celecoxib induced cell death. Innovation and ConclusionThese novel findings for direct effects of celecoxib on mitochondria to induce metastatic cancer cell death via a ROS-dependent pro-oxidative mechanism provide supportive evidence for its combinatorial use as a chemosensitizing agent complementing chemotherapies to improve response rates in patients with advanced metastatic cancers.

Role of cyclin-dependent kinase 4/6 inhibitors in the current and future eras of cancer treatment.

Cyclin-dependent kinase 4/6 inhibitors, which act by inhibiting progression from the G1 to S phases of the cell cycle, include palbociclib, ribociclib, abemaciclib, and trilaciclib. Palbociclib and ribociclib are currently food and drug administration-approved for use in combination with aromatase inhibitors in postmenopausal women with metastatic hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. Palbociclib is also food and drug administration-approved for use in combination with fulvestrant in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer progressing after endocrine therapy. Abemaciclib is the newest cyclin-dependent kinase 4/6 inhibitor to gain Food and Drug Administration (FDA) approval, specifically as monotherapy for hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer previously treated with chemotherapy and endocrine therapy. Abemaciclib also shares a similar indication with palbociclib for use in combination with fulvestrant in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer progressing after endocrine therapy. Trilaciclib use remains largely investigational at this time. However, despite FDA-approval for only metastatic hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer, all four cyclin-dependent kinase 4/6 inhibitors have shown promise in hematologic malignancies and non-breast solid tumors. Although further research is needed, cyclin-dependent kinase 4/6 inhibitors represent intriguing developments in the treatment of various malignancies, including those with such poor prognoses as glioblastoma multiforme, mantle cell lymphoma, and metastatic melanoma. We discuss the approved indications, current research, and areas of future exploration for palbociclib, ribociclib, abemaciclib, and trilaciclib.

Cost-Effectiveness of Second-Line Endocrine Therapies in Postmenopausal Women with Hormone Receptor-positive and Human Epidermal Growth Factor Receptor 2-negative Metastatic Breast Cancer in Japan.

Exemestane (EXE), exemestane + everolimus (EXE + EVE), toremifene (TOR), and fulvestrant (FUL) are second-line endocrine therapies for postmenopausal hormone receptor-positive (HR +)/human epidermal growth factor receptor 2-negative (HER2 -) metastatic breast cancer (mBC) in Japan. Although the efficacy of these therapies has been shown in recent studies, cost-effectiveness has not yet been determined in Japan. This study aimed to examine the cost-effectiveness of second-line endocrine therapies for the treatment of postmenopausal women with HR + and HER2 - mBC. A Markov model was developed to analyze the cost-effectiveness of the therapies over a 15-year time horizon from a public healthcare payer's perspective. The efficacy and utility parameters were determined via a systematic search of the literature. Direct medical care costs were used. A discount rate of 2% was applied for costs and outcomes. Subgroup analysis was performed for non-visceral metastasis. A series of sensitivity analyses, including probabilistic sensitivity analysis (PSA) and threshold analysis were performed. Base-case analyses estimated incremental cost-effectiveness ratios (ICERs) of 3 million and 6 million Japanese yen (JPY)/quality-adjusted life year (QALY) gained for TOR and FUL 500mg relative to EXE, respectively. FUL 250mg and EXE + EVE were dominated. The overall survival (OS) highly influenced the ICER. With a willingness-to-pay (WTP) threshold of 5 million JPY/QALY, the probability of TOR being cost-effective was the highest. Subgroup analysis in non-visceral metastasis revealed 0.4 and 10% reduction in ICER from the base-case results of FUL5 500mg versus EXE and TOR versus EXE, respectively, while threshold analysis indicated EVE and FUL prices should be reduced 73 and 30%, respectively. As a second-line therapy for postmenopausal women with HR +/HER2 - mBC, TOR may be cost-effective relative to other alternatives and seems to be the most favorable choice, based on a WTP threshold of 5 million JPY/QALY. FUL 250mg is expected to be as costly and effective as EXE. The cost-effectiveness of EXE + EVE and FUL 500mg could be improved by a large price reduction. However, the results are highly sensitive to the hazard ratio of OS. Policy makers should carefully interpret and utilize these findings.

Impairment of the Gnα11-controlled expression of claudin-1 and MMP-9 and collective migration of human breast cancer MCF-7 cells by DHEAS

Although dehydroepiandrosterone sulfate (DHEAS) constitutes the most abundant steroid in humans, in-depth investigations of its effects are rather scarce. We address here DHEAS effects on the estrogen receptor-positive metastatic human breast cancer cell line MCF-7. We focus on DHEAS-mediated signaling that might influence expression of claudin-1 and matrix metalloproteinase-9 (MMP-9), both known to be critical factors for migration and invasiveness of various cancers, including breast cancer cells.Physiological concentrations of DHEAS trigger persistent phosphorylation of Erk1/2 in MCF-7 cells. Exposure of these cells for 24 h to 1 μM DHEAS also leads to a significant reduction of claudin-1 expression that cannot be prevented by high concentrations of the steroid sulfatase inhibitor STX64, indicating that desulfation and further conversion of DHEAS to some other steroid hormone is not required for this action. In addition, exposure of MCF-7 cells to the same concentration of DHEAS completely abolishes MMP-9 expression and considerably impairs cell migratory behavior.Abrogation of Gnα11 expression by siRNA prevents the stimulatory effect of DHEAS on Erk1/2 phosphorylation, consistent with a G-protein-coupled receptor being involved in the DHEAS-induced signaling. Nevertheless, Gnα11 also has direct effects that do not depend on DHEAS; thus, when Gnα11 expression is suppressed, expression of claudin-1 and MMP-9 as well as cell migration are significantly reduced.This is the first report demonstrating direct involvement of DHEAS and Gnα11 in the regulation of claudin-1 and MMP-9 expression and migration of MCF-7 cells.

Regulation of OPN expression in MDA‐MB‐435: role of genistein

Breast cancer is the most common cause of cancer‐related mortality in women worldwide and metastatic spread is the leading cause of breast cancer deaths. Osteopontin (OPN) plays a functional role in normal mammary gland development, progression of breast cancer and in malignancy of breast cancer. High expression of OPN is observed in high‐grade metastatic malignant human gliomas and in the highly invasive and metastatic human breast cancer cell lines (e.g. MDA‐MB‐435). Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor‐β (ERβ), is known to have antitumoural properties and also induce apoptotic cell death of ERα‐negative breast cancer cells via p53‐dependent pathway. The isoflavone has been reported to exert antiproliferative activity in some breast cancer cell lines; however, its role with respect to OPN expression is not well understood. Therefore, to address this, the expression level of OPN was investigated upon genistein treatment in MDA‐MB‐435. The cells were treated with different concentrations of genistein for different time intervals; the expression of OPN, the signaling pathways and the promoter regions involved in the regulation of OPN were studied. Genistein displayed an inhibitory effect on OPN protein and mRNA levels in a dose dependent manner and treatment with 50 μM genistein for 24 h showed to be promising. Analysis of the signaling pathways showed no activation of Akt/PKB pathway, no alteration in p‐MEK½ expression whilst an increase in the expression of p‐ERK½ was observed in genistein treated group. These results indicate that genistein acts as an anti‐metastatic agent by inhibiting OPN expression through the activation of p‐ERK½ in MDA‐MB‐435 cells. The transcription factors responsible for this event are being studied in details.Support or Funding InformationDBT (GoI) No. BT/253/NE/TBP/2011 dated May 07, 2012This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P6-14-05: Phase 2 study evaluating the efficacy and safety of eribulin mesylate administered biweekly for patients with human epidermal growth factor receptor 2-negative metastatic breast cancer

Abstract Background: Eribulin mesylate, a microtubule inhibitor, is approved in the US for the treatment of patients (pts) with metastatic breast cancer (MBC) who have previously received at least 2 chemotherapeutic regimens for the treatment of metastatic disease, including an anthracycline and a taxane. The recommended dose is 1.4 mg/m2 (equivalent to 1.23 mg/m2 eribulin [expressed as free base]) on day (D) 1 and D8 of a 21-D cycle. However, this schedule can result in dose delays and reductions due to myelosuppression. A dosing regimen of eribulin (1.4 mg/m2) administered intravenously (IV) biweekly (Q2W; on D1 and D15) in 28-D cycles was evaluated with the intent of improving eribulin's safety profile without compromising efficacy. Methods: Female pts with human epidermal growth factor receptor (HER)2-negative MBC, who had received 2-5 prior chemotherapy regimens and had ECOG PS ≤2 were enrolled in 12 sites in the US. Prophylactic granulocyte colony-stimulating factor (G-CSF) was not allowed. If neutropenia occurred, growth factors were used during eribulin treatment at the physician's discretion. Primary endpoints were objective response rate (ORR) and disease control rate (DCR). Secondary endpoints were progression-free survival (PFS), overall survival (OS), dose intensity (measured by feasibility rate), safety and tolerability. Results: Median age of the 58 enrolled pts was 64 yrs (range: 38-85). The majority of pts (93%) had ECOG PS ≤1, and 12% of pts had triple-negative MBC. Number of prior chemotherapeutic regimens: 2 (17% of patients), 3 (24%), 4 (27%), and 5 (31%). 76% Of pts had visceral disease and 86% had previous taxane therapy. ORR (95% confidence interval [CI]) was 12% (5-24), DCR (CR+PR+SD) was 65% (95% CI: 51-77), and CBR (CR+PR+SD ≥23 weeks) was 30% (95% CI: 18-43) [n=57]. Median PFS (95% CI) was 3.6 mo (2.9-4.1). Median OS (95% CI) was 13.2 mo (10.6-not estimable). 6-Month and 12-month OS rates were 84% and 54%, respectively. Dose intensity measured by the feasibility rate (defined as the percentage of pts completing the first 2 and 4 cycles without a dose delay >5 days or dose reduction due to an adverse event [AE]) was 70% and 46%, respectively. The most frequent AEs (all grades) were neutropenia (69%), fatigue (48%), alopecia (45%), and constipation (36%). 22% Of pts had grade (G) 1 alopecia and 22% of pts had G2 alopecia. 72% Of pts had G3/4 AEs: neutropenia, 57%, and peripheral neuropathy, 12%. G3 peripheral sensory neuropathy occurred in 9% of pts, with no G4 incidence. There were 2 deaths (1 sepsis, 1 acute respiratory failure), which were considered not related to treatment. 50% (29/58) Of all patients received at least 1 dose of growth factor and 70% (28/40) of patients with neutropenia received growth-factor support. Conclusions: Tumor response rates and OS of this treatment schedule in a heavily pretreated patient population were similar compared to previously reported phase 3 studies of eribulin. The toxicities associated with biweekly eribulin were manageable. Citation Format: Smith II J, Irwin A, Jensen L, Tedesco K, Misir S, Zhu W, Almonte A, He Y, Olivo M, O'Shaughnessy J. Phase 2 study evaluating the efficacy and safety of eribulin mesylate administered biweekly for patients with human epidermal growth factor receptor 2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-14-05.

A Cancer Cell-Selective and Low-Toxic Bifunctional Heterodinuclear Pt(IV)-Ru(II) Anticancer Prodrug.

Although different types of metal-based anticancer complexes have been synthesized, novel complexes to reduce the serious side effect of cisplatin and conquer cancer metastasis are still highly desired. Here, we report the synthesis, characterization, and biological activity of a novel heterodinuclear Pt(IV)-Ru(II) anticancer prodrug. The Pt(IV)-Ru(II) complex exhibits good stability in both water and PBS solution. Biological evaluation revealed that this bifunctional Pt(IV)-Ru(II) complex utilizes the advantages of two metal centers to have both cytotoxicity and antimetastatic property as designed. Although the complex has comparable cytotoxicities to cisplatin in tested cancer cell lines, this prodrug selectively kills cancer but not normal cells, and the IC50 values of the Pt(IV)-Ru(II) complex are 7-10 times higher than those of cisplatin toward normal cells. The cancer cell selectivity is further demonstrated by a cancer-normal cell coculture system. In addition, the antimetastatic properties of the heterodinuclear complex are assessed by using highly metastatic human breast cancer cells, and the results show that the migration and invasion of cancer cells are effectively restrained after the treatment. Moreover, the Pt(IV)-Ru(II) complex displays lower toxicity than cisplatin in developing zebrafish embryos. We, therefore, report an example of heterodinuclear Pt(IV)-Ru(II) complex not only to defeat both drug resistance and cancer metastasis but also having significantly improved cancer cell selectivity and reduced in vivo toxicity than cisplatin.

Co-delivery of docetaxel and doxorubicin using biodegradable PEG-PLA micelles for treatment of breast cancer with synergistic anti-tumour effects

ABSTRACTPoly(ethylene glycol)-poly(lactic acid) copolymer, prepared by ring opening polymerization, was used as a single platform to co-deliver both hydrophilic doxorubicin and hydrophobic docetaxel (DTX) in a simulated physiological environment. The average size of the negatively charged drug loaded polymeric micelles were found to be 293 nm. The drug loading (%) and encapsulation efficiency (%) were calculated to be 1.21 and 59.0, respectively. The in vitro cytotoxicity test using MCF7 breast cancer cells was conducted using 1 × 104 cells in 10% FBS and 1% antibiotic, and the absorbance of formazan was evaluated at 570 nm. Cell growth inhibition by MTT assay showed viability of 33% of the MCF7 cells after treatment with drug-loaded micelles for 48 h. Controlled release of drugs from the polymeric micelles indicated a burst release effect initially; whereas, 98% of drug could be released at pH 7.4 within a time period of 96 h. Time period for drug release shorten to 48 h only in simulated mild acidic pH (5.4) condition. The in vitro drug release study from micelles indicated synergistic cytotoxicity effect in human metastatic breast cancer MCF7 cell.

Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media.

GRP78 (an Mr 78 kDa calcium dependent glucose binding protein) is located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER-/PR-/HER2+ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER-/PR-/HER2-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Nonmetastatic estrogen receptor positive (ER+) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/mL) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A) or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

The HER2 inhibitor lapatinib potentiates doxorubicin-induced cardiotoxicity through iNOS signaling.

Rationale: Lapatinib (LAP) is a crucial alternative to trastuzumab upon the onset of drug resistance during treatment of metastatic human epidermal growth factor receptor 2-positive breast cancer. Like trastuzumab, LAP is commonly used alongside anthracyclines as a combination therapy, due to enhanced anti-cancer efficacy. However, this is notably associated with cardiotoxicity so it is imperative to understand the mechanisms driving this cardiotoxicity and develop cardioprotective strategies. To this end, here we utilize human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), which exhibit several characteristics representative of in vivo cardiomyocytes that make them breakthrough models to study drug toxicity.Methods: We investigated LAP- and doxorubicin (DOX)-induced toxicity in hPSC-CMs and evaluated the involvement of inducible nitric oxide (NO) synthase (iNOS). The significance of iNOS-mediated cardiotoxicity was furthermore evaluated in animal studies.Results: LAP synergistically increased DOX toxicity in hPSC-CMs in a dose- and time-dependent manner. At concentrations that were otherwise non-apoptotic when administered separately, LAP significantly potentiated DOX-induced hPSC-CM apoptosis. This was accompanied by increased iNOS expression and pronounced production of NO. iNOS inhibition significantly reduced hPSC-CM sensitivity to LAP and DOX co-treatment (LAP-plus-DOX), leading to reduced apoptosis. Consistent with our observations in vitro, delivery of an iNOS inhibitor in mice treated with LAP-plus-DOX attenuated myocardial apoptosis and systolic dysfunction. Moreover, inhibition of iNOS did not compromise the anti-cancer potency of LAP-plus-DOX in a murine breast cancer xenograft model.Conclusions: Our findings suggest that iNOS inhibition is a promising cardioprotective strategy to accompany HER2-inhibitor/anthracycline combination therapies. Furthermore, these results support the promise of hPSC-CMs as a platform for investigating cardiotoxicity and developing cardioprotectants as a whole.

Phosphorylated S6 Kinase-1 as Predictive Marker of Lapatinib Efficacy in Human Epidermal Growth Factor Receptor 2-Positive Metastatic Breast Cancer Patients

Phosphorylated S6 Kinase-1 as Predictive Marker of Lapatinib Efficacy in Human Epidermal Growth Factor Receptor 2-Positive Metastatic Breast Cancer Patients

KISS1 tumor suppressor restricts angiogenesis of breast cancer brain metastases and sensitizes them to oncolytic virotherapy in vitro

KISS1 tumor suppressor protein regulates cancer cell invasion via MMP9 metalloproteinase. Downregulation of KISS1 gene expression promotes progression of breast cancer and melanoma, resulting in the development of distant metastases. In the current study, we investigated whether restoration of KISS1 expression in KISS1-deficient human metastatic breast cancer cells holds potential as an advanced anticancer strategy. To this end we engineered an infectivity-enhanced conditionally-replicative human adenovirus type 5 encoding KISS1 as an “arming” transgene in the Ad5 E3 region for an ectopic KISS1 expression in transduced cancer cells. The oncolytic potential of the vector was examined using brain-invading metastatic clones of CN34 and MDA-MB-231 breast cancer cells, which supported high levels of AdKISS1 replication, correlating with a robust CRAd-mediated cytotoxicity. Secretion of cellular factors responsible for tumor angiogenesis, cell-to-cell communication and anti-tumoral immune responses upon KISS1 expression in breast cancer cells was analyzed by a RayBiotech Kiloplex Quantibody array. Overall, our results indicate that KISS1 transgene expression provides an important benefit for CRAd-mediated cytotoxicity in breast cancer cells and holds potential as an anticancer treatment in conjunction with oncolytic virotherapy of breast and other metastatic cancers.

Protein Acyltransferase DHHC3 Regulates Breast Tumor Growth, Oxidative Stress, and Senescence.

DHHC-type protein acyltransferases may regulate the localization, stability, and/or activity of their substrates. In this study, we show that the protein palmitoyltransferase DHHC3 is upregulated in malignant and metastatic human breast cancer. Elevated expression of DHHC3 correlated with diminished patient survival in breast cancer and six other human cancer types. ZDHHC3 ablation in human MDA-MB-231 mammary tumor cell xenografts reduced the sizes of both the primary tumor and metastatic lung colonies. Gene array data and fluorescence dye assays documented increased oxidative stress and senescence in ZDHHC3-ablated cells. ZDHHC3-ablated tumors also showed enhanced recruitment of innate immune cells (antitumor macrophages, natural killer cells) associated with clearance of senescent tumors. These antitumor effects were reversed upon reconstitution with wild-type, but not enzyme-active site-deficient DHHC3. Concomitant ablation of the upregulated oxidative stress protein TXNIP substantially negated the effects of ZDHHC3 depletion on oxidative stress and senescence. Diminished DHHC3-dependent palmitoylation of ERGIC3 protein likely played a key role in TXNIP upregulation. In conclusion, DHHC3-mediated protein palmitoylation supports breast tumor growth by modulating cellular oxidative stress and senescence. Cancer Res; 77(24); 6880-90. ©2017 AACR.

In Vivo Detection of HSP90 Identifies Breast Cancers with Aggressive Behavior.

Purpose: Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers.Experimental Design: We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye. HS131 uptake into cells correlated with cell membrane expression of Hsp90 and was used to image molecular subtypes of murine and human breast cancers in vitro and in murine models.Results: HS131 imaging was both sensitive and specific in detecting the murine 4T1 breast cancer cell line, as well as subclones with differing metastatic potential. Highly metastatic subclones (4T07) had high HS131 uptake, but subclones with lower metastatic potential (67NR, 168FARN) had low HS131 uptake. We generated isogenic cell lines to demonstrate that overexpression of a variety of specific oncogenes resulted in high HS131 uptake and retention. Finally, we demonstrated that HS131 could be used to detect spontaneous tumors in MMTV-neu mice, as well as primary and metastatic human breast cancer xenografts. HS131 could image invasive lobular breast cancer, a histologic subtype of breast cancer which is often undetectable by mammography.Conclusions: An HSP90-targeting nIR probe is sensitive and specific in imaging all molecular subtypes of murine and human breast cancer, with higher uptake in aggressive and highly metastatic clones. Clinical studies with Hsp90-targeting nIR probes will be initiated shortly. Clin Cancer Res; 23(24); 7531-42. ©2017 AACR.

Clinical pattern of primary systemic therapy and outcomes of estrogen receptor-positive, HER2-negative metastatic breast cancer: a review of a single institution.

In the management of estrogen receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer (ER+HER2-MBC) patients, endocrine therapy (ET) is preferred to chemotherapy (CT) as a primary systemic therapy (PST) when tumor burden is not high. However, there are no definite criteria for choosing a PST, transitioning from ET to CT or using maintenance ET subsequent to CT. We reviewed the medical records of 311 ER+HER2-MBC patients who underwent CT from September 2002 to December 2016 and assessed their outcomes. Of the 311 patients, 178 (57%) received ET as a PST (ET-first group), and 133 (43%) received CT prior to ET (CT-first group). The ET-first group showed a median overall survival (OS) from the diagnosis of MBC (OSMBC) of 1593days, and the median OS from the initiation of CT (OSCT) was 938days. Patients with visceral involvement, liver metastasis, soft tissue metastasis, ≥3 organ involvement, or primary advanced BC at the MBC diagnosis showed a significantly higher tendency to be assigned to the CT-first group (P<0.01 for any visceral involvement, P<0.05 for all others). Maintenance ET was available in 74 (55.6%) patients in the CT-first group, who showed a significantly better OSMBC and OSCT than patients without maintenance ET (median OSMBC 1423 and 867days, respectively, P<0.0001; median OSCT 1350 and 637days, respectively, P<0.0001). Our findings suggest the possibility for changing the treatment paradigm of patients with ER+HER2-MBC, so a randomized prospective study is warranted to determine the optimum sequence of systemic therapies.

Ultrasound-mediated drug delivery by gas bubbles generated from a chemical reaction

Highly echogenic and ultrasound-responsive microbubbles such as nitrogen and perfluorocarbons have been exploited as ultrasound-mediated drug carriers. Here, we propose an innovative method for drug delivery using microbubbles generated from a chemical reaction. In a novel drug delivery system, luminol encapsulated in folate-conjugated bovine serum albumin nanoparticles (Fol-BSAN) can generate nitrogen gas (N2) by chemical reaction when it reacts with hydrogen peroxide (H2O2), one of reactive oxygen species (ROS). ROS plays an important role in the initiation and progression of cancer and elevated ROS have been observed in cancer cells both in vitro and in vivo. High-intensity focussed ultrasound (HIFU) is used to burst the N2 microbubbles, causing site-specific delivery of anticancer drugs such as methotrexate. In this research, the drug delivery system was optimised by using water-soluble luminol and Mobil Composition of Matter-41 (MCM-41), a mesoporous material, so that the delivery system was sensitive to micromolar concentrations of H2O2. HIFU increased the drug release from Fol-BSAN by 52.9 ± 2.9% in 10 minutes. The cytotoxicity of methotrexate was enhanced when methotrexate is delivered to MDA-MB-231, a metastatic human breast cancer cell line, using Fol-BSAN with HIFU. We anticipate numerous applications of chemically generated microbubbles for ultrasound-mediated drug delivery.

Abstract 5890: Osteoblasts are educated into a tumor-associated stromal cell by disseminated breast cancer cells and mediate breast cancer cell proliferation in the bone microenvironment

Abstract Breast cancer has a predilection for bone metastasis, where the five-year survival rate is bleak. Disseminated breast cancer cells invade bone and can remain undetectable and untreatable for decades during a period of reduced proliferation. Our work reveals that osteoblasts are educated into a tumor-associated stromal cell by disseminated breast cancer cells and alter their production of exosomal microRNAs that regulate cancer cell cycle. Osteoblasts were grown to various stages of maturity and incubated with the conditioned medium (CM) from human breast cancer cells to produce tumor-associated osteoblasts (TAOs). TAOs and their CM were characterized, then TAO cells admixed with human breast cancer cells in-vitro or in-vivo to elucidate the impact of TAOs on the tumor microenvironment. Osteoblasts undergo an inflammatory stress response and gain tumor-associated stromal cell-like properties when either co-cultured or treated with the CM of human breast cancer cells. These changes were mediated by crosstalk via gap junction intercellular communication (connexin 43) and exosome exchange that occurred between TAO cells and human breast cancer cells. TAO-derived exosomes were found to contain increased amounts of microRNAs 320a and 193b compared to normal osteoblasts or breast cancer cells. microRNA 320a is associated with decreased cellular proliferation and induction of G0 phase of the cell cycle. miR 193b has been shown to regulate cyclin D1 expression and repress cellular proliferation. Knock-down of miRs 320a and 193b in breast cancer cells resulted in a reduced number of cancer cells in G0 and increased numbers of cancer cells in G1/S/G2/M phases of the cell cycle. Furthermore, TAO cell CM led to decreased activation of TRAP+ osteoclasts. When TAO cells were admixed with human breast cancer cells and inoculated in-vivo in mice, tumors grew more slowly and were at least 50% smaller than tumors composed of normal osteoblasts plus breast cancer cells, or breast cancer cells inoculated alone. Mice inoculated with an admix of TAO cells plus human metastatic breast cancer cells lived ~20 days longer than mice inoculated with an admix of normal osteoblasts plus metastatic breast cancer cells, or breast cancer cells alone. These data also suggest that TAO cells regulate the proliferation of metastatic breast cancer cells in the tumor microenvironment. Overall, these date suggest that osteoblasts are an important source of factors, specifically exosomal microRNAs, in breast cancer bone metastasis. The nature of these factors suggest their importance for facilitating disseminated breast cancer cell proliferation, as well as osteoclast activation in the bone microenvironment. Supported by NIH 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; (NRSA) T32 CA079448, NIH 3 R00 CA178177 for KMB. Citation Format: Frank Marini, Julia Chifman, Janet Tooze, Candelaria Gomez-Manzano, Karen M. Bussard. Osteoblasts are educated into a tumor-associated stromal cell by disseminated breast cancer cells and mediate breast cancer cell proliferation in the bone microenvironment [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 5890. doi:10.1158/1538-7445.AM2017-5890

Abstract 2197: Characterization of immunoliposomes as delivery vehicles for the Rac inhibitor Ehop-016 in HER2 type breast cancer

Abstract Due to the critical need for improved therapeutics against advanced Stage IV breast cancer, we developed and pre-clinically validated the small molecule Ehop-016 as a potential anti-metastatic drug. Ehop-016 is a small molecule inhibitor of the interaction Rac with the guanine nucleotide exchange factor (GEF) Vav2, which consequently decreases invasive actin structures and cellular migration. In a human epidermal growth factor receptor 2 (HER2) type breast cancer model in nude mice, Ehop-016 inhibits mammary tumor growth, angiogenesis, and metastasis. Moreover, Ehop-016 reduces HER2++ cancer cell viability and migration, individually or in combination with anti HER2 or EGFR directed therapeutics. By inhibiting the key signaling intermediate Rac, Ehop-016 has the potential to block signaling downstream of HER2 and EGFRs, as well as signaling via the myriad of receptors that activate Rac, and inhibit cancer progression. Our new results demonstrate that Ehop-016 inhibits both cancer cells and leukocytes in the tumor microenvironment, as well as cytokine levels (IL-6) in mouse plasma. Even though these characteristics are desirable in anticancer agents, systemic therapy with Ehop-016 may adversely affect normal immune system function. Therefore, a strategy was developed for the targeted delivery of Ehop-016 into mammary tumors, using nanoliposomes containing the current breast cancer therapeutic, Trastuzumab, a monoclonal antibody directed at the HER2. We tested various nanoliposome formulations as targeted delivery systems for Ehop-016. First, the encapsulation efficiency of Ehop-016 loaded liposomes was tested using different drug loading methods. To obtain HER2 targeted liposomes, we prepared Trastuzumab-lipid conjugates and incorporated them into the Ehop-016 containing liposomes. The binding, internalization and selectivity of our HER2 targeted delivery system was verified in human metastatic breast cancer cell lines, by using fluorescence microscopy to assess the co-staining of the lipid bilayer and Trastuzumab. In order to evaluate the effectiveness and selectivity of the immunoliposomes in delivering Ehop-016 and inhibiting Rac activity, pull-down assays were performed to detect active Rac-GTP in cells treated with Ehop-016 alone or Trastuzumab-conjugated liposomes with Ehop-016. Results show that the Trastuzumab-conjugated liposomes are more selective at binding to HER2+ breast cancer cells and inhibiting Rac, than treatment with Ehop-016 alone. In summary, our novel delivery system containing Ehop-016 and Trastuzumab has been shown to be effective in breast cancer cells, and needs to be validated in animal models of metastatic breast cancer. The development of this targeted delivery system has implications, not only in the field of breast cancer therapeutics, but may be adapted for the treatment of other diseases, and used to overcome the side effects of current treatments. Citation Format: Michael J. Rivera-Robles, Pablo E. Vivas-Mejia, Linette Castillo-Pichardo, Cornelis Vlaar, Eliud Hernández, Suranganie Dharmawardhane. Characterization of immunoliposomes as delivery vehicles for the Rac inhibitor Ehop-016 in HER2 type breast cancer [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 2197. doi:10.1158/1538-7445.AM2017-2197