Search

Introduction. Breast cancer recurrence can develop years after primary treatment. Crosstalk between breast cancer cells and their stromal microenvironment may influence tumor progression. Our primary study aim was to determine whether endothelin-1 (ET-1) expression in tumor and stroma predicts breast cancer relapse. The secondary aim was to determine ET-1/endothelin receptor A (ETAR) role on signaling pathways and apoptosis in breast cancer. Experimental Design. Patients with histologically documented stages I–III invasive breast cancer were included in the study. ET-1 expression by immunohistochemistry (IHC) in tumor cells and stroma was analyzed. Association between ET-1 expression and clinical outcome was assessed using multivariate Cox proportional hazard model. Kaplan-Meier curves were used to estimate disease-free survival (DFS). In addition, the effect of ET-1/ETAR on signaling pathways and apoptosis was evaluated in MCF-7 and MDA-MB-231 breast cancer cells. Results. With a median followup of 7 years, ET-1 non-enriched tumor phenotype had a significant association with favorable disease-free survival (HR=0.16; 95% CI 0.03–0.77; P value Conclusions. We found ET-1 expression in tumor and stroma to be an independent prognostic marker for breast cancer recurrence. Prospective studies are warranted to examine whether ET-1 expression in tumor/stroma could assist in stratifying patients with hormone receptor positive breast cancer for adjuvant therapy.

Pancreatic adenocarcinoma is associated with advanced presentation and poor survival. Currently approved therapies have minimal effect on patient survival. Pancreatic adenocarcinomas have a high incidence of activated K-RAS, which may confer resistance to epidermal growth factor receptor (EGFR) inhibitors. Mixed lineage kinase-3 (MLK3) is a MAP3K that activates multiple MAPK pathways. The role of MLK3 in the pathophysiology and resistance to therapy of pancreatic adenocarcinoma has not been investigated. MLK3 is over expressed in pancreatic cancer cell lines compared to an immortalized pancreatic epithelial cell line. The requirement of MLK3 for cell proliferation and survival of pancreatic cancer cell lines, PANC-1 and MiaPaCa-2, was investigated using RNA interference (siRNA) and MLK inhibitor, K252a, alone or in conjunction with the EGFR inhibitor, Compound 56. Ablation of expression of MLK3 via siRNA-mediated gene silencing and pharmacological inhibition of MLK3 by K252a each decreased cell viability in both pancreatic cancer cell lines, with a concurrent decrease in the activation of ERK, JNK and AKT. Concomitant inhibition of EGFR and MLK3 induced apoptosis, as evidenced by increased cleavage of PARP and caspase-3. These results suggest that MLK3 plays an important role in survival and proliferation of pancreatic cancer cell lines and that inhibition of MLK3 may enhance the therapeutic efficacy of EGFR inhibitors in the treatment of pancreatic cancer.

e23156 Background: Previous studies demonstrated the efficacy of HDACi and Pi as single agents in BC. The synergistic combination of HDACi and Pi has been shown to be effective in clinical studies in other tumor types, including multiple myeloma, non-small cell lung cancer, and pancreatic cancer. In this in vitro study, we explored the hypothesis that combination of these two agents may improve overall antitumor effect in BC. Methods: In the experiment, we used 3 BC cell lines: MCF7 (ER+), MDA-MB-231 (ER-), and BCSV (isolated from metastatic ER+ tumor which lost ER expression with disease progression). The cells were treated with carfilzomib (Pi) and scriptaid (HDACi) as single agents and in combination. Cell viability was determined using MTT assay, and combination indexes were determined using CalcuSyn software. Additionally, cells were cultured in 3D tumor spheroid model, and response to treatment was assessed on day 5, 10, 15 and 20. Results: In comparison with control, single agent HDACi, single agent Pi and combination HDACi/Pi increased cell viability by 6%, decreased cell viability by 60% and by 69% respectively (p < 0.0001) in MCF7 cells; and decreased cell viability by 25%, 59% and 66% respectively (p = 0.0061) in BCSV cells. The combination indexes for most HDACi and Pi combinations were lower than 1.0, indicating synergistic effects in MCF-7 and BCSV cells but not in MDA-MB-231 cells. In 3D cultures, the combination of carfilzomib 1µM and scriptaid 10µM led to significant reduction of tumor spheroid size as compared to single agent. By day 20, BCSV tumor spheroid size increased by 258% in control group and by 50% in Pi, decreased by 17% in HDACi and by 30% in HDACi/Pi combination group (p < 0.0001). Conclusions: Combination of HDACi and Pi exhibited synergistic activity in both MCF7 and BCSV monolayer cultures. Additionally, combination HDACi and Pi significantly decreased 3D tumor spheroid size as compared to either single agent alone. Further study is needed to evaluate the mechanisms of Pi/HDACi combination in estrogen-mediated BC tumorigenesis.

Pharmacological studies on Oltipraz [4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione)] were conducted with normal healthy subjects using various doses and schedules. Administration of single doses (1, 2 and 3 mg/kg) resulted in detectable drug levels in the serum (mean peak serum concentrations 16, 61 and 205 ng, respectively) and urine. The t1/2 was short (4.4, 4.1 and 5.3 hours respectively) and no steady state was achieved after multiple daily doses for 12 days. Introduction of a loading dose during the first day produced a steady state when 1.5 and 2.0 mg/kg/day were used. Daily administration of Oltipraz sustained the steady state with insignificant variations. Consumption of a high fat diet increased the serum and urine concentrations of Oltipraz (30-60%) compared to the low fat diet. Two subjects experienced flatulence during the administration of the drug. One subject developed numbness and pain in the thumbs with occurrence of small purplish-black spots resembling those observed in subacute endocarditis. These changes disappeared 10 days after discontinuation of the drug. No changes in peripheral blood counts, biochemical profile or thyroid function tests were observed after four weeks of Oltipraz. Further studies with a larger number of healthy subjects are needed for clarification of the safety and biological efficacy of small doses of Oltipraz during chronic administration.

Associations between stress hormones and preterm delivery have not been fully explored. In this study, pregnant women enrolled from 52 clinics in 5 Michigan communities (1998–2004) provided urine samples for 3 days (waking and bedtime) during midpregnancy. Urinary catecholamine levels (epinephrine, norepinephrine, and dopamine) were measured in a subcohort (247 preterm and 760 term deliveries), and a 3-day median value was calculated. Polytomous logistic regression models assessed relations between catecholamine quartiles (of the median) and a 4-level outcome variable (i.e., term (referent) and 3 preterm delivery subtypes: spontaneous; premature rupture of membranes; and medically indicated). Final models incorporated other relevant covariates (e.g., creatinine, demographic, behavior). The risk of spontaneous preterm delivery was increased in the highest versus lowest quartile of norepinephrine and dopamine: norepinephrine, waking (adjusted odds ratio (AOR) ¼ 3.7, 95% confidence interval (CI): 1.8, 7.9) and bedtime (AOR ¼ 2.5, 95% CI: 1.3, 4.9); dopamine, waking (AOR ¼ 2.6, 95% CI: 1.4, 5.1) and bedtime (AOR ¼ 2.3, 95% CI: 1.2, 4.6). Adjusted odds ratios were further strengthened after removing women whose placentas showed evidence of acute infection or vascular pathology. High catecholamine levels in maternal urine may be indicative of excess stressors and/or predisposition to elevated sympathetic activation that contributes to increased risk of spontaneous preterm delivery. catecholamines; dopamine; epinephrine; gestational age; norepinephrine; pregnancy; pregnancy outcome; premature birth

e22221 Background: Targeted therapies for breast cancer patients carrying BRCA-1/2 mutations are emerging. Histone deacytelase inhibitors (HDACi) represent a promising class of drugs for the treatm...

The Pregnancy Outcomes and Community Health (POUCH) Study enrolled women in their 15th to 27th week of pregnancy from 52 prenatal clinics located in five communities. After the study began, an at-home protocol to measure maternal stress was added, which included collection of urine and saliva twice a day (waking = AM, bedtime = PM) for three consecutive days, and completion of a daily diary. The at-home protocol was in place for 2852 POUCH participants at enrollment, and 81.3% (n = 2318) consented and returned samples and diary. For these analyses, salivary cortisol was analysed in a subset of 846 women who delivered at term. Day-to-day correlation coefficients for AM cortisol (0.45-0.55) exceeded those for PM cortisol (0.31-0.43). Study diaries indicated that there was variation in the time interval between waking and sample collection. Analyses of discrete intervals showed the same awakening response pattern in cortisol levels that has been reported in studies with serial AM sampling. The adjusted mean AM cortisol (microg/dL) was 0.506 at 0-15 min post-waking, 0.544 at 16-30 min (P0.05), 0.582 at 31-60 min (P0.01), and 0.515 at60 min post-waking. In addition, adjusted mean AM cortisol in samples collected at or before 9 a.m. was higher than that in samples collected after 9 a.m. (0.564 vs. 0.510 microg/dL, P0.01). Among working women, adjusted mean AM cortisol was higher on work days than non-work days (0.564 vs. 0.489 microg/dL, P0.01), and in multiparae compared with primiparae (0.551 vs. 0.502 microg/dL, P = 0.07). The parity effect was not evident in non-working women. The adjusted mean PM cortisol significantly increased as week of pregnancy at sampling increased, but was not significantly related to time of collection, work day and parity. Factors influencing AM and PM cortisol levels in pregnancy appear to differ. In studies of AM cortisol levels, it is important to gather data on time of sample collection, interval from waking to sample collection, parity and work.

Background: Endothelin-1 (ET-1) and endothelin A receptor (ETAR) are implicated in breast cancer growth and progression. ET-1 is secreted by both tumor cells and stroma (macrophages, endothelial cells). The purpose of this study is to evaluate ET-1/ETAR role on cell proliferation and its effects on EGFR signaling pathway. Materials and Methods: Two breast cancer cell lines: MCF-7 and MDA-MB-231 were stimulated with ET-1. Proliferation of breast cancer cells was analyzed using MTT assay. Protein expression (EGFR, pEGFR, AKT, pAKT, ERK, pERK) was evaluated by Western blot. siRNA knockdown of endothelin A receptor (ETAR) was performed in MCF-7 and MDA-MB-231. Results: ET-1 stimulated proliferation in both cell lines. Interestingly, at higher ET-1 concentrations cell proliferation was more pronounced in MCF-7 when compared with MDA-MB-231. Stimulation with ET-1 activated EGFR and downstream signaling proteins (pAKT and pERK) in both cell lines. Incubation of breast cancer cells with ET-1 for 48 hours had different effect on total EGFR level: 3 fold increase of total EGFR was seen in MCF-7, while no change was detected in MDA-MB-231. siRNA against ETAR decreased ET-1 induced cell proliferation and reduced total EGFR level in MCF-7. Conclusions: These observations suggest that ET-1/ETAR plays an important role in survival and proliferation of breast cancer cells. However, those effects are diverse in different subtypes of breast cancer. Our results may represent an improved selective targeted treatment strategy, especially for estrogen receptor positive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-03-06.

Rationale: Currently approved therapies, including EGFR inhibitors, have minimal effect on survival in pancreatic cancer (PC). HSP90 is a molecular chaperone that specifically enables activity of numerous protein kinases, including EGFR and other signaling proteins. We have reported that simultaneous inhibition of EGFR and HSP90 may be additive in inhibiting cell viability in PC cell lines. Here we report possible molecular mechanisms underlying HSP90 and EGFR inhibition. Methods: MiaPaCa-2 and PANC-1 were treated with EGFR inhibitor, compound 56 (CMP56) (5 − 35 μmol), and HSP90 inhibitor geldanamycin (250 nmol − 1μmol). Growth inhibition was evaluated using the MTT assay, and apoptosis was evaluated by caspase-3 and PARP cleavage. The effect on relevant cell signaling proteins was assessed by immunoblotting with specific antibodies directed against EGFR and effectors such as AKT, STAT-3 and JNK. Results: Growth inhibition was significantly (p < 0.05) higher in both MiaPaCa-2 and PANC1 cells treated with CMP56 and geldanamycin than in cells treated with either agent at various concentrations. The molecular effects are shown in table 1. There was a decrease in phosphorylation of EGFR in bothe cell lines after treatment with CMP56. Concomitant inhibition of EGFR and HSP90 was associated with decreased phosphorylation of ERK, JNK and AKT and with an increase in caspase-3, PARP cleavage in MiaPaCa-2 cells, but not in PANC-1 cells. Conclusions: These results suggest that HSP90 inhibition in MiaPaCa-2 cells induces down regulation of EGFR, STAT-3 and AKT, but not in PANC-1 cells. Higher concentrations of geldanamycin may be required in PANC-1cells. Combined inhibition of EGFR and HSP90 induced apoptosis additively. We are studying further to understand the other potential molecular pathways involved in this strategy.Table.Molecular effects of EGFR and HSP90 inhibition in PC Cell lines ↘ - Decrease, - No change, GEL - Geldanamycin (500 nmol), CMP-56 - Compound 56 (10 μmol)Protein of interest MiaPaCa-2 PANC-1 GELCMP56GEL + CMP56 GELCMP56GEL + CMP56EGFR↘-↘↘ --↘AKT↘-↘↘ --↘STAT-3↘-↘↘ --↘ Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1657.

6076 Background: More efficacious treatments of head and neck squamous cancers (HNSCC) are urgently needed. HNSCC overexpress EGFR, yet the efficacy of EGFR inhibitors is marginal, with minimal effect on patient survival. Mixed lineage kinases (MLKs) are members of a large family of mitogen-activated protein kinase (MAPK) kinase kinases (MAPKKKs) that activate MAPK pathways, involving JNK, ERK 1/2 and p38 cell signaling pathways. (Gallo KA, Johnson GL, Nat Rev Mol Cell Biol. 2002;3:663–72) We explored the role of MLK3 in HNSCC as a possible therapeutic target. Methods: The requirement of MLK3 for cell proliferation and survival of HNSCC cell lines UM-SCC-38 and -47 (HPV+) (obtained from TE Carey, U Michigan) was investigated using RNA interference (siRNA) and MLK3 inhibitor K252a to inhibit the expression and activity of MLK3. Cells were also exposed to EGFR inhibitor Compound 56 at various concentrations. Proliferation was assessed with MTT after 72 h exposure. The effect of MLK3 inhibition on relevant cell signaling pathways was assessed by immunoblotting with activation-specific phosphoantibodies directed against MAPKs, the prosurvival kinase Akt and apoptotic proteins. Results: MLK3 is overexpressed in malignant HNSCC cell lines compared to normal tonsil lysate, and its expression can be inhibited by siRNA or K 252a. Inhibition of MLK3 expression is associated with downregulation of phospho-Akt and with decreased proliferation. Compound 56 inhibits proliferation at lower concentrations in HPV+ cell line UM-SCC-47 (10 microM) compared with UM-SCC-38 (25 microM), inhibits expression of pErk, and decreases expression of pAkt. Combining siRNA or K252a and compound 56 showed less pAkt expression and increased apoptosis (caspase 3 or PARP cleavage) compared with compound 56 alone. Conclusions: MLK3 may have a role in avoiding apoptosis in HNSCC cells. Its inhibition may enhance the effects of EGFR inhibition in HNSCC. No significant financial relationships to disclose.