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Her2 positive (Her2+) breast cancer (BC) accounts for 18-20% of all breast cancer subtypes and is associated with high risk of death. Trastuzumab, the first Food and Drug Administration-approved targeted therapy for BC, represents a key milestone in the personalized treatment of Her2+ metastatic disease (Her2+ MBC). However, the median duration of response is less than a year, due to either primary or acquired resistance to the therapy. In addition, there is currently no conclusive biomarker for the response of patients to trastuzumab. Therefore, understanding the development of resistance to trastuzumab is of our interest. Recent studies have proposed various potential mechanisms leading to the resistance, including p27 rapid degradation. Previously, our study and other research demonstrated that Jab1 degrades p27 in breast cancer. These findings suggest that Jab1 over-expression contributes to trastuzumab resistance by facilitating p27 degradation. Jab1/CSN5 (c-Jun activation domain-binding protein 1) is over-expressed in 50% of primary and 90% of metastatic breast cancers, while its expression is low or absent in normal adult breast tissues. We previously identified that high expression of Jab1 is associated with shorter progression-free survival in breast cancer patients. In this study, our preliminary data showed that the knockdown of Jab1 sensitizes the breast cancer cells to trastuzumab treatment in a dose-dependent manner. Mechanistically, we found that Jab1 over-expression is significantly correlated with the activation of Akt pathway in Her2+ breast cancer cells and xenograft model. Interestingly, activated Akt, due to PTEN loss or PI3K activating mutation has been widely implicated in the potential mechanism to trastuzumab resistance. Therefore, our results suggest that targeting Jab1 overcomes the resistance to trastuzumab via interfering with PI3K/Akt pathway. In general, our study identifies Jab1 as a novel contributor to trastuzumab resistance and elucidates its potential mechanisms of actions. The successful completion of the study can potentially be translated to the clinic for the benefit of patients refractory to the therapy. Also our study suggests Jab1 expression level can be used as a predictive marker for trastuzumab treatment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-02.

The platinum-acridine anti-cancer agent [PtCl(en)(LH)](NO(3))(2) (1) (en = ethane-1,2-diamine, LH = N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide, acridinium cation) and the clinical drug cisplatin were studied in chemoresistant non-small cell lung cancer (NSCLC) cell lines for their cytotoxic potency and cell-kill mechanisms. In the three cell lines tested (NCI-H460, NCI-H522, and NCI-H1435) compound 1 shows a pronounced cytotoxic enhancement of 40-200-fold compared to cisplatin at inhibitory concentrations reaching the low-nanomolar range. Based on changes in cell adhesion and cell morphology, monitored in real time by impedance measurements, compound 1 kills NCI-H460 cells significantly more efficiently than cisplatin at equitoxic concentrations. Flow cytometry analysis of NCI-H460 cells reveals a robust S-phase arrest of cells treated with compound 1, whereas cells treated with cisplatin progress to G2/M of the cell cycle. A pronounced inhibition of DNA replication in 75% of viable cells is observed in NCI-H460 cells treated with compound 1 at an IC(90) molar concentration for 48 h, based on the reduced incorporation of the fluorophore-clickable nucleoside analogue 5-ethynyl-2´-deoxyuridine (EdU) into newly synthesized DNA. The distinct cell-cycle perturbations and cell-kill potential of compound 1 are discussed in the light of the DNA interactions of this agent and its potential to overcome cisplatin resistance in NSCLC.

An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86-93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependent cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.

Context.—African American women with breast cancer have worse prognoses than non–African Americans and might benefit with a race-specific prognostic marker such as PTEN (phosphatase and tensin homologue), a tumor suppressor protein. Reduced PTEN expression is associated with worse outcomes and resistance to trastuzumab in human epidermal growth factor receptor 2–positive breast cancers. Standardized PTEN evaluation is therefore needed. Objective.—To assess PTEN as a race-specific prognostic marker in breast cancer by using a novel semiquantitative score and a percent staining assessment. Design.—Between 1991 and 1996, 146 patients with invasive ductal adenocarcinoma were grouped by race and recurrence; there was a median follow-up of 7.2 years with 63 recurrences. Immunostaining of PTEN in tissue microarrays was correlated with race, recurrence, node positivity, stage, size, age, estrogen/progesterone receptor status, grade, and DNA ploidy. Results.—No significant racial difference was detected in mean PTEN values using either the semiquantitative score (P = .46) or the percent staining (P = .54). Unrelated to race, the percentage of tumor cells with positive PTEN expression correlated with longer time to recurrence (P = .047), positive estrogen receptor status (P = .009), and lower tumor grade (P = .005). The semiquantitative score correlated with positive estrogen receptor status (P = .01) and lower tumor grade (P = .001). Conclusions.—PTEN expression is not a race-specific biologic prognostic marker for invasive ductal adenocarcinoma. Increased PTEN expression correlates with longer time to recurrence, positive estrogen receptor status, and lower tumor grade. The novel semiquantitative score may be used to evaluate PTEN expression, but the approximate percentage of tumor cells with any PTEN staining may be the most useful measure of PTEN expression.

Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.

19-Nor-1α,25-dihydroxyvitamin D 2 (paricalcitol) is an analogue of 1,25(OH) 2 D 3 with reduced calcemic effects that is approved in the United States for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has anticancer activity in prostate cancer cells. We tested the effects of paricalcitol on the HL-60 leukemia cells, studying cellular differentiation, cell cycle changes, apoptosis and cellular proliferation. Paricalcitol at 10 −8 M concentration induced the maturation of HL-60 cells in a time-dependent manner, as shown by increased expression of CD11b differentiation surface antigen. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was markedly increased after exposure to paricalcitol at 10 −8 M for 72 h. Paricalcitol inhibited colony formation of HL-60 cells in a soft agar semisolid media after 10-day incubation (estimated IC 50 of 5×10 −9 M. Exposure to 10 −8 M paricalcitol for 72 h increased the number of cells in G 0 /G 1 phase, and decreased the number of cells in S phase, and significantly increased the number of HL-60 cells undergoing apoptosis. The concentration required to achieve inhibition of growth of HL-60 cells is comparable to clinically achievable levels. These findings support the clinical evaluation of paricalcitol as an antileukemia agent.

Laminin-1 is a basement membrane glycoprotein implicated in tumor-host adhesion, which involves the cell-binding domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are necessary for binding to laminin-1 have not been characterized. To identify this laminin-binding oligosaccharide sequence, GlcNSO4-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) residues were isolated from human breast cancer cell (MCF-7)-derived HS through hydrazinolysis/high pH (4.0) nitrous acid treatment/[3H]NaBH4 reduction. These oligosaccharides were chromatographed on a laminin-1 affinity column. A high affinity dodecasaccharide was isolated and characterized. Disaccharide analysis yielded IdoA(2-SO4) → AManR(6-SO4) as the only disaccharide upon treatment of this dodecasaccharide with nitrous acid at low pH (1.5). The sequence of laminin-binding high affinity oligosaccharide is therefore [IdoA(2-SO4) → GlcNSO4(6-SO4)]5[IdoA(2-SO4) → AManR(6-SO4)]. Low affinity dodecasaccharides composed of [IdoA(2-SO4) → GlcNSO4(6-SO4)]5, [IdoA(2-SO4) → GlcNSO4] were also isolated by laminin-1 affinity chromatography. Molecular modeling studies indicate that a heparin-binding peptide sequence corresponding to amino acid residues 3010–3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of laminin-1, modeled as a right-handed α-helix, carries an array of basic residues well placed to bind to clusters of sulfate groups on the high affinity dodecasaccharide.

This study was undertaken to evaluate the deoxyribonucleic acid content and S-phase fraction in advanced epithelial ovarian carcinomas to determine whether lymph node metastases are biologically distinct from peritoneal sites of metastases.Thirty-five patients with stage III or IV epithelial ovarian cancer who had undergone complete pelvic and paraaortic lymphadenectomy had representative samples from the primary ovarian tumor, peritoneal metastases, and lymph node metastases analyzed by flow cytometry for deoxyribonucleic acid nuclear content and S-phase fraction.Diploid cell lines are found in metastatic lymph nodes (52%) significantly more frequently than in peritoneal metastases (25%, p0.02) or in primary ovarian tumors (26%, p0.001). The ploidy category frequency distribution of peritoneal metastases mirrors that found in the primary tumor, and both are significantly different from the ploidy category frequency distribution found in metastatic lymph nodes. Heterogeneity among sites is common, being identified in 54% of patients. Peritoneal metastases are more likely to be concordant with the primary tumor (69%) than are lymph node metastases (39%, p0.001). Mean S-phase fraction did not differ overall by site but was significantly different between diploid and aneuploid samples by site. Diploid lymph node metastases were found to have the lowest mean S-phase fraction (7.2% +/- 3.3%), and aneuploid lymph node metastases had the highest mean S-phase fraction (22.3% +/- 10.2%). Diploidy of the primary tumor is a positive predictor of long-term survival. Tumoral heterogeneity and lymph node metastases are not related to survival in this group of patients who underwent therapeutic pelvic and aortic lymphadenectomy.A high proportion of tumor deposits found in metastatic lymph nodes are diploid with a low S-phase fraction. Therapeutic pelvic and aortic lymph node dissection removes disease that, on the basis of flow cytometric characteristics, may be predicted to be resistant to chemotherapy and radiation therapy.

BACKGROUND Since the discovery of nm23 (nonmetastatic) by Steeg et al. in 1988, a number of tumor cohort studies have shown an inverse relationship between the levels of expression of the nm23-H1 protein and disease aggressiveness and tumor metastatic potential. METHODS The relationship between the expression of nm23 protein and the metastatic potential of human breast carcinoma was analyzed in cell lines, xenografts, and in a retrospective lymph node negative breast carcinoma population. The lymph node negative breast carcinoma study was comprised of 40 patients: 19 with nonrecurrent and 21 with recurrent disease. The 40 patients were matched according to age, cathepsin D, tumor size, percent S-phase, DNA ploidy, steroid receptor status, and tumor grade. Nm23-H1 protein levels in cell lines and xenografts were analyzed quantitatively using Western blot analyses and semiquantitatively in tissue sections using immunocytochemistry. Immunocytochemical analysis of lymph node negative breast tumors was graded as the percent of tumor staining positive for nm23 and the intensity of staining. The metastatic potentials of the cell lines and xenografts were assessed as the ability to form metastatic lesions in nude mice. In the lymph node negative breast carcinoma patients, the metastatic potential was characterized as the incidence of breast carcinoma recurrence. RESULTS The MCF-7 cell line expressed four- and tenfold higher levels of nm23-H1 than the highly metastatic MDA-MB-435 and MDA-MB-231 cells, respectively. Among the xenografts and cell lines, there was an inverse correlation between nm23-H1 expression and metastatic potential in athymic nude mice (correlation coefficient [R] = -0.51). The differences between the levels of nm23-H1 among the metastatic and nonmetastatic cell lines and xenografts were not statistically significant. Statistical analyses indicated that neither the intensity nor the percent of tumor staining positive for nm23 expression was correlated to the recurrence of breast carcinoma in the lymph node negative patient population that had been matched for other clinical prognostic markers. CONCLUSIONS There was an inverse correlation (R = -0.51) between the levels of nm23-H1 expression in cell lines and xenografts and the metastatic potential in nude mice. In the retrospective lymph node negative breast carcinoma population, no clear association was demonstrated between the expression of nm23 and breast carcinoma recurrence. This observation suggests the nm23 expression does not predict outcome in lymph node negative breast carcinoma patients. Cancer 1997; 79:1158-65. © 1997 American Cancer Society.

This report describes a companion flow cytometry study (Cancer and Leukemia Group B (CALGB)--8869) using tumors derived from patients enrolled in a large randomized clinical trial (CALGB-8541) performed on 1,572 patients with early stage, node-positive breast cancer. The CALGB initiated an adjuvant breast cancer trial in 1985 to determine if dose intensification (dose of drug per unit time) of chemotherapy was related to relapse-free and overall survival. Patients were randomized by pretreatment clinical variables to one of three different dosage regimens of chemotherapy. Using a tumor enrichment procedure, 442 paraffin-embedded blocks were analyzed by flow cytometry, and S-phase fraction (SPF) was analyzed by three different methods. Ploidy analysis was performed using standard procedures. Tissue from 90% of the patients was suitable for ploidy analysis, whereas only 68% could be assessed for SPF. With a median follow-up time of 80 months, our results show that ploidy status had no clinical utility, whereas high SPF predicted poorer overall survival. The rectangular fit model for SPF was more predictive of outcome than both the area fit model and a computer fit model (modfit) for SPF. In univariate analysis, patients with a low SPF (10%) had a better prognosis than those patients with a high SPF (10%), but they responded equally well to the different treatment regimens. Patients with high SPF (10%) had longer relapse-free and overall survival to high dose chemotherapy compared to low or standard dose chemotherapy. Multivariate analysis indicated that treatment intensity as well as the number of positive nodes, tumor size, steroid receptor status, and c-erb B-2 expression were significant in predicting overall and disease-free survival. The multivariate analysis, however, revealed that SPF was significant in predicting overall but not disease-free survival, but there was no longer any relationship among SPF, dose intensity, and outcome.

VEGF, an endothelial-specific mitogen, is an important tumor angiogenesis growth factor. The major receptor for VEGF on endothelial cells is KDR. We hypothesized that an intrabody could bind newly synthesized KDR and block receptor transport to the cell surface, thereby inhibiting important VEGF effects. We expressed a single chain antibody (p3S5) to KDR with or without the endoplasmic reticulum (ER) retention signal (KDEL), using either a plasmid (p3S5-HAK) or a tet-off adenoviral system (Ad-HAK). Plasmid-mediated expression of the tethered intrabody significantly reduced KDR expression (from 82.5+/-12.5% to 27.9+/-13.6% of cells; P0.01) and thymidine incorporation in successfully transfected cells. Ad-HAK infection resulted in intrabody expression in90% of human umbilical vein endothelial cells (HUVECs), producing marked (80%) apoptosis at 48 h postinfection. The intrabody was essential for these effects, as confirmed by inhibiting its expression with doxycycline or by expressing irrelevant genes (lacZ, GFP). Cell death was dependent on KDR, because Ad-HAK infection of cell lines with minimal or no KDR had little effect on cell viability. Infected HUVECs were unable to form tubes on Engelbreth Holm-Swarm (EHS) tumor gel matrix. These results demonstrate the potential for development of an intrabody-based strategy to block angiogenesis and prevent tumor growth.

Use of the antibody trastuzumab to kill HER2+ breast cancer cells is an attractive therapy because of its specificity and minimal adverse effects. However, a large fraction of HER2+ positive patients are or will become resistant to this treatment. No other markers are used to determine sensitivity to trastuzumab other than HER2 status.Using the xCELLigence platform and flow cytometry, we have compared the ability of mononuclear cells (MNCs) from normal and breast cancer patients to kill different breast cancer cell lines in the presence (i.e., ADCC) or absence of trastuzumab. Image analysis and cell separation procedures were used to determine the differential contribution of immune cell subsets to ADCC activity. The assay demonstrated that ADCC activity is dependent on the presence of trastuzumab, the level of HER2 expression on the target, and the ratio of MNCs to tumor cells. There is a wide range of ADCC activity among normal individuals and breast cancer patients for high and low HER2-expressing tumor targets. Fresh MNCs display higher ADCC levels compared with cryopreserved cells. Natural killer cells display the highest ADCC followed by monocytes. T cells and B cells were ineffective in killing. A major mechanism of killing of tumor cells involves insertion of granzyme B and caspase enzymes via the antibody attached MNCs.

This is the consensus statement regarding the clinical utility of DNA cytometry in breast cancer from the DNA Cytometry Consensus Conference held in Prout's Neck, Maine, USA, on October 1–4, 1992. Guidelines for clinical DNA cytometry generated at that meeting also appear in this issue of Breast Cancer Research and Treatment.

Sclerostin domain containing 1 (SOSTDC1) protein regulates processes from development to cancer by modulating activity of bone morphogenetic protein (BMP) and wingless/int (Wnt) signaling pathways. As dysregulation of both BMP and Wnt signaling has been observed in breast cancer, we investigated whether disruption of SOSTDC1 signaling occurs in breast cancer. SOSTDC1 mRNA expression levels in breast tissue were examined using a dot blot. Affymetrix microarray data on SOSTDC1 levels were correlated with breast cancer patient survival using Kaplan–Meier plots. Correlations between SOSTDC1 protein levels and clinical parameters were assessed by immunohistochemistry of a breast cancer tissue microarray. SOSTDC1 secretion and BMP and Wnt signaling were investigated using immunoblotting. We found that SOSTDC1 is expressed in normal breast tissue and this expression is reduced in breast cancer. High levels of SOSTDC1 mRNA correlated with increased patient survival; conversely, SOSTDC1 protein levels decreased as tumor size and disease stage increased. Treatment of breast cancer cells with recombinant SOSTDC1 or Wise, a SOSTDC1 orthologue, demonstrated that SOSTDC1 selectively blocks BMP-7-induced Smad phosphorylation without diminishing BMP-2 or Wnt3a-induced signaling. In conclusion, SOSTDC1 mRNA and protein are reduced in breast cancer. High SOSTDC1 mRNA levels correlate with increased distant metastasis-free survival in breast cancer patients. SOSTDC1 differentially affects Wnt3a, BMP-2, and BMP-7 signaling in breast cancer cells. These results identify SOSTDC1 as a clinically important extracellular regulator of multiple signaling pathways in breast cancer.

Ara-U-induced S-phase accumulation and the interaction between high concentrations of ara-U (HiCAU) and ara-C were investigated in L1210 leukemia cells in vitro. Treatment of exponentially growing L1210 murine leukemia cells with ara-U (200-1000 microM) for 48 h caused a dose-dependent accumulation of cells in the S-phase. The extent of this ara-U-induced S-phase accumulation correlated with ara-U incorporation into DNA and with increases of up to 172% and 464% in the specific activities of deoxycytidine kinase and thymidine kinase, respectively, over control values. Metabolism of 1 microM ara-C following the exposure of cells to ara-U (1 mM) resulted in 4.5 pmol araC DNA/mg protein vs 2.1 pmol/mg protein in control cells. Although 48-h exposure of cells to 200 and 400 microM ara-U is not cytotoxic, it enhances the cytotoxicity of ara-C (10-100 microM) 4- to 10-fold. Ara-U-induced S-phase accumulation is inhibited by deoxypyrimidine nucleosides but not by pyrimidine or deoxypurine nucleosides. Some of the ara-U and ara-C concentrations used in this study are achievable in clinical practice, and ara-U/ara-C interactions may explain in part the unique therapeutic utility of high-dose ara-C.

Nuclear and membrane markers that have been related to proliferative activity were measured by flow cytometry. The markers studied were transferrin receptor (TR), Ki-67 antigen, and epidermal growth factor receptor (EGFR). Two-color analysis for DNA via propidium iodide binding and for antigen expression via either a direct or indirect immunofluorescence assay was performed on three different cell lines and a solid human tumor model. The three cell lines tested were MCF-7 (breast), K-562 (leukemia), and A431 (a squamous cell). The solid tumor was obtained by subcutaneous injection of A431 cells into an athymic nude mouse. Our results demonstrate that TR are cell-cycle specific and can be readily measured in the cell lines. Ki-67 antigen is also cell-cycle specific in the cell lines tested, but the mean channel specific fluorescence uptake varies in the cell types. Finally, the EGFR was observed only in the A431 cell line, with most cells equally expressing this receptor. A bimodal distribution of EGFR was observed in A431 cells obtained from a solid tumor grown in an athymic nude mouse system. This suggests that cell line analysis may not always represent what might be observed under in vivo conditions. There are advantages to flow cytometry measurements of these factors which might be useful in predicting how patients should be treated and possibly the prognosis of cancer patients.

Characterization of breast cancer cells by histology, flow cytometry, and steroid receptors was performed on 197 Stage I1 breast node positive cancer patients given adjuvant chemotherapy, plus tamoxifen for patients with positive hormone receptors. Histologic and steroid receptor assays were performed using standard techniques; flow cytometric analysis was performed from paraffin-embedded blocks obtained from the primary tumor. Quality control studies on reproducibility, tissue heterogeneity, and analysis procedures have been included. Of the 197 patients studied, aneuploidy was found in 102 (52%); the median %S value was 8% with a range of 0.4% to 38%. Our results demonstrated that number of positive nodes, receptor status, and grade were of prognostic value. Cell cycle kinetic data were not of independent prognostic value in this series. However, ploidy could differentiate in prognosis in the receptor-negative subgroup. Patients with receptor-negative tumors had a significantly better overall survival if the tumor was diploid in nature. Cell kinetics was not significantly prognostic for either receptor subgroup, although patients with higher %S tended to have better relapse-free and overall survival. This is in disagreement with other studies and may demonstrate that treatment has confounded our results and diminished the ability of flow cytometry data to help predict outcome. Cancer 66:1810-1816,1990. INCE ITS DEVELOPMENT, there has been great interest S in using flow cytometry to predict the clinical course of patients with breast cancer.'-3 Several groups have compared ploidy and cell kinetic measurements to other clinical parameters including age, menopausal status, receptor content, tumor size, histologic type and grade, and nodal status. There is general consensus that aneuploid

The effects of estradiol (E), medroxyprogesterone acetate (MPA), and tamoxifen (TAM) on the growth of a human ovarian carcinoma cell line, BG-1, were evaluated using a tumor clonogenic assay (HTCA). BG-1 contains significant quantities of estrogen and progesterone receptors. Growth inhibition by TAM and growth stimulation by MPA were demonstrated using continuous drug exposure. Estradiol resulted in a marginal increase in colony formation. With each of these three drugs, the greatest response occurred in the larger colonies (generally greater than or equal to 60 microns). Combinations of each of these three steroidal agents with three different cytotoxic drugs were studied in the HTCA. Synergistic activities were produced with TAM combined with either cisplatin or doxorubicin. Additive effects were seen with TAM and cyclophosphamide. Although predominantly additive or synergistic, the effects were variable with MPA and all three cytotoxic agents. Combinations of estradiol with cytotoxic agents were no more active than the cytotoxics alone. These findings indicate a biological rationale for hormonal manipulation as therapy in ovarian cancer.

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 microM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [3H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.

Iron is critical for cell growth and proliferation. Iron chelators are being explored for a number of clinical applications, including the treatment of neurodegenerative disorders, heart disease, and cancer. To uncover mechanisms of action of tachpyridine, a chelator currently undergoing preclinical evaluation as an anticancer agent, cell-cycle analysis was performed. Tachpyridine arrested cells at G2, a radiosensitive phase of the cell cycle, and enhanced the sensitivity of cancer cells but not nontransformed cells to ionizing radiation. G2 arrest was p53 independent and was accompanied by activation of the checkpoint kinases CHK1 and CHK2. G2 arrest was blocked by UCN-01, a CHK1 inhibitor, but proceeded in CHK2 knock-out cells, indicating a critical role for CHK1 in G2 arrest. Tachpyridine-induced cell-cycle arrest was abrogated in cells treated with caffeine, an inhibitor of the ataxia-telangiectasia mutated/ataxia-telangiectasia-mutated and Rad3-related (ATM/ATR) kinases. Further, G2 arrest proceeded in ATM-deficient cells but was blocked in ATR-deficient cells, implicating ATR as the proximal kinase in tachpyridine-mediated G2 arrest. Collectively, our results suggest that iron chelators may function as antitumor and radioenhancing agents and uncover a previously unexplored activity of iron chelators in activation of ATR and checkpoint kinases.

Background. African-American women (AAW) have more adverse tumor characteristics than non-African American women (non-AAW) and there is a need for a specific predictive marker for AAW recurrence. Tumors with higher grade and proliferative activity are associated with overexpression of the glucose transporter (Glut-1). An examination of Glut-1 expression relative to recurrence and no recurrence was conducted on both groups to determine if it predicts prognosis and could predict a race specific prognosis. Methods. A breast cancer data set containing clinical information including race and biological characteristics was generated between 1991 and 1996. Tissue samples were selected from this group with similar characteristics in both racial groups for a retrospective analysis of Glut-1 expression using tissue microarrays (TMAs). Mean Glut-1 expression for intensity and percent of tumor cells staining was determined using standard immunohistochemistry. Results. Clinical and biological differences were noted in the original data set between AAW and non-AAW. No significant difference between races was noted in mean Glut-1 values using a subset which had similar characteristics. The mean Glut-1 did not predict recurrence but a rounded score did indicate that higher levels of Glut-1 expression was indicative of a lower disease free survival (p = 0.063). The actual average mean for AAW with no recurrence was significantly lower than any other group (p = 0.04). Conclusions. TMA analysis for Glut-1 expression may be useful to predict disease free survival but it does not predict race specific recurrence.

Polyploidy is a profound phenotype found in tumors and its mechanism is unknown. We report here that when B-cell lymphoma gene-2 (Bcl-2) was overexpressed in a Chinese hamster ovary cell line that was deficient in CTP:phosphocholine cytidylyltransferase (CT), cellular DNA content doubled. The higher DNA content was due to a permanent conversion from diploid cells to tetraploid cells. The mechanism of polyploid formation could be attributed to the duplication of 18 parental chromosomes. The rate of conversion from diploid to tetraploid was Bcl-2 dose dependent. The diploid genome was not affected by Bcl-2 expression or by CT deficiency alone. Endogenous CT or expression of recombinant rat liver CTα prior to Bcl-2 expression prevented the formation of polyploid cells. This conversion was irreversible even when both initiating factors were removed. In this study, we have identified Bcl-2 as a positive regulator and CTα as a negative regulator of polyploid formation.

MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-gamma. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARgamma independently of this receptor.

Background: Despite years of research, it is still unclear which women with node-negative (N–) breast cancer will need adjuvant chemotherapy and which women are being treated unnecessarily. Our goal was to determine which factors best predicted disease free survival (DFS) or cancer-specific overall survival (OS) and, therefore, select the correct patients for treatment. A total of 11 parameters were measured: estrogen receptor (ER), progesterone receptor (PR), age, race, ploidy status, %G0/G1 (% non-DNA synthesis), %S (% S-phase), cathepsin D status, size, stage, and histologic grade. Results: In this prospective study, we followed 556 N– patients diagnosed between 1991 and 1996. The tumors were 56% ER, 51% PR, 30% diploid, with a mean %S of 8.9%. The level of cathepsin D ranged from 0.50 to 155 pmol/mg of protein with a mean of 42.9 pmol/mg of protein. There were 87 recurrences (16%) and 72 cancer deaths (13%), with a median follow-up of 7.8 years. Ploidy status (p 0.01), S-phase activity (p 0.003), G1 phase activity (p 0.02) and age (p 0.01) were able to significantly predict DFS in a univariate manner. All of the measurable factors were significant or borderline significant in predicting OS in a univariate manner except for age, race, and ER status. In multivariate analysis with S-phase included, it was the only remaining factor in DFS and OS; with S-phase excluded, age and ploidy status remained as factors for DFS in stepwise regression, while PR, size, and cathepsin D were the remaining factors that predicted cancer-specific OS. The effect of adjuvant treatment on prognosis was also analyzed. Conclusions: Both biochemical and clinical parameters have the potential to predict prognosis for N– breast cancer. In this large prospective clinical trial, with a median follow-up of 7.8 years, no individual marker adequately predicted the prognosis for an individual patient. %S activity was the best independent marker, but only 77% of the tumors provided this value. Subset analysis provided improved prognostication, but there were limits to its utility. These data represents a definitive study starting in 1991 and ending in 2002. © 2004 Wiley-Liss, Inc.

Background Herceptin, a humanized antibody to HER-2, is now utilized in the clinic for metastatic breast cancer treatment. The response rate for HER-2+ patients is only 30% and little is known as to mechanisms of resistance. The mechanism of Herceptin action is also unknown but has been related to cell cycle inhibition. Methods The effects of Herceptin and other antibody treatments were determined by cell counting and cell cycle analysis. HER-2 and p27 expression levels were analyzed by flow cytometry and levels of activated AKT were compared by Western blot analysis. Cellular HER-2 and p27 expression was measured by immunofluorescence. Results Herceptin treatment of BT-474 cells results in inhibition of cell growth and arrest in the G1 phase. The efficacy of growth arrest was not directly correlated to the binding affinity of antibodies to Her-2. Our laboratory has developed cell lines that are resistant to Herceptin treatment. In resistant cell lines, binding of antibodies is not hindered. However, Herceptin has completely lost the ability to inhibit cell proliferation. Yet, the mouse isotype 4D5 maintains significant inhibitory activity upon Herceptin-resistant clones. Conclusions Herceptin binds effectively to Her-2 on the cell surface of Herceptin-resistant cell lines and the level of Her-2 expression on the cell surface is not downregulated. Herceptin resistance is not due to downregulation of levels of AKT protein expression, although, phosphorylation of AKT is enhanced in resistant lines and could have a role in resistance. Resistance appears to correlate with the loss of nuclear expression of the cyclin-dependent kinase inhibitor, p27, as defined by immunofluorescence and flow cytometry studies and cdk-2 binding studies. © 2004 Wiley-Liss, Inc.

Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens. The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells. HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control. MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells. Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity. Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance. Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors. Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively. We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function. These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.

We performed studies to test synergism be- tween the growth inhibitory effects of genistein and vitamin D compounds on prostatic epithelial cells. Isobolographic analysis demonstrated that genistein, in combination with the hormonally active form of cholecalciferol, 1,25-dihy- droxycholecalciferol, synergistically inhibited the growth of primary human prostatic epithelial cells (HPEC) and pros- tate cancer cells. Synergistic growth inhibition of HPEC was also observed between genistein and the low-calcemic vitamin D compound 25-hydroxycholecalciferol. Flow cy- tometry with HPEC indicated that genistein induced arrest in the G2M phase, whereas 1,25-dihydroxycholecalciferol or 25-hydroxycholecalciferol induced arrest in the G1/0 phase of the cell cycle. Combining genistein with either vitamin D compound resulted in both G2M and G1/0 arrest in HPEC. In contrast, flow cytometry of prostate cancer cells indicated that both genistein and 1,25-dihydroxycholecal- ciferol induced a G1/0 arrest either alone or in combination. These are the first studies that demonstrate synergism between the prostatic cell growth inhibition elicited by genistein and that elicited by vitamin D compounds. J. Nutr. 132: 3191-3194, 2002.

19-Nor-1α,25-dihydroxyvitamin D2 (paricalcitol) is an analogue of 1,25(OH)2D3 with reduced calcemic effects that is approved for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has recently been reported to have anticancer activity in prostate cancer. In order to explore paricalcitol as a potential agent against leukemia, we tested its effects on HL-60 and U937 leukemia cell lines. We studied cellular differentiation via expression of CD11b and CD14 surface antigens using flow cytometry, and via the nitroblue tetrazolium (NBT) assay. Cell cycle was analyzed using propidium iodide staining. Apoptosis was assessed with the annexin V assay. Cellular proliferation was determined via colony inhibition on semisolid medium. Paricalcitol induced the maturation of HL-60 and U937 cells, as shown by increased expression of CD11b differentiation surface antigen. CD14 showed increased expression in HL-60 but not in U937 cells. After exposure to paricalcitol at 10−8 M for 72 h, the ability of HL-60 cells to reduce NBT was markedly increased. Conversely, U937 cells were unchanged. Paricalcitol inhibited colony formation of both HL-60 and U937 cell lines in semisolid medium after a 10-day incubation (estimated IC50 of 3×10−8 M in HL-60 cells and 4×10−8 M in U937 cells). Paricalcitol at 10−8 M and 10−7 M caused a significant dose- and time-dependent increase of apoptosis in HL-60 cells (P

Herceptin is a humanized antibody that binds to the product of the HER-2 oncogene. Clinical studies have indicated that treatment with Herceptin may slow disease progression in tumors expressing high levels of the HER-2 antigen. However, the mechanism of this action is not known.Four different cell lines were used that had different levels of HER-2 expression. Treated and nontreated cells were analyzed for DNA strand breaks and cell cycle perturbation using standard flow cytometry methods.In this study we found that cell lines expressing high levels of HER-2, when treated with Herceptin, exhibited marked increases in DNA strand breaks as measured by the TUNEL assay, and that these cells also exhibited slowed growth. BT-474 and SKBR-3 cell lines, both of which express high levels of the HER-2 antigen, had significant increases in labeled nucleotide expression at 3 and 6 day time points following exposure to Herceptin at a concentration of 10 microg/ml. Similar treatment of MCF-7 and MDA-231 cell lines, both of which express low levels of HER-2, had little effect on the level of labeled nucleotide expression at either the 3 or 6 day time points. Following 4 days of Herceptin treatment, BT-474 and SKBR-3 cell lines had significant decreases in the percentage of cells in the S phase of growth. This effect was not seen in either the MCF-7 or MDA-231 cell lines.Herceptin has a biological effect only on cells that contain high levels of HER-2. This effect is a decrease in cell proliferation that is coincident with, and may be caused by an increase frequency of DNA strand breaks.

Background We have previously reported that high expression of the erbB-2 gene (also known as HER-2/neu and ERBB2) in breast cancer is associated with patient response to dose-intensive treatment with cyclophosphamide, doxorubicin (Adriamycin), and 5-flurouracil (CAF) on the basis of short-term follow-up of 397 patients (set A) with axillary lymph node-positive tumors who were enrolled in Cancer and Leukemia Group B (CALGB) protocol 8541. Methods To validate those findings, we conducted immunohistochemical analyses of erbB-2 and p53 protein expression in an additional cohort of 595 patients (set B) from CALGB 8541, as well as a molecular analysis of erbB-2 gene amplification in tumors from all patients (sets A and B). Marker data were compared with clinical, histologic, treatment, and outcome data. Results Updated analyses of data from set A (median follow-up, 10.4 years) showed an even stronger interaction between erbB-2 expression and CAF dose, by use of either immunohistochemical or molecular data. A similar interaction between erbB-2 expression and CAF dose was observed in all 992 patients, analyzed as a single group. However, for set B alone (median follow-up, 8.2 years), results varied with the method of statistical analysis. By use of a proportional hazards model, the erbB-2 expression-CAF dose interaction was not significant for all patients. However, in the subgroups of patients randomly assigned to the high- or the moderate-dose arms, significance was achieved. When patient data were adjusted for differences by use of a prognostic index (to balance an apparent failure of randomization in the low-dose arm), the erbB-2 expression-CAF dose interaction was significant in all patients from the validation set B as well. An interaction was also observed between p53 immunopositivity and CAF dose. Conclusions The hypothesis that patients whose breast tumors exhibit high erbB-2 expression benefit from dose-intensive CAF should be further validated before clinical implementation. Interactions between erbB-2 expression, p53 expression, and CAF dose underscore the complexities of predictive markers where multiple interactions may confound the outcome.

The aim of this project is to determine the role of a novel antioncogene (nm23) in the spread of cancer. We propose that nm23 prevents metastatic spread of disease by down regulation of proteolytic activity that is necessary for tumor cells to migrate to other parts of the body. We have developed two methods for measurement of nm23 in tumor cells and they are comparable. We have set up test for the cathepsin D and the other components of the urokinase plasminogen activator system. We have measured the expression of these components in 3 cell lines, 8 primary cultures grown in nude mice and more than 50 human primary breast tumors. The relationships of these proteolytic components and nm23 expression are in progress. We have prepared a new selection vector containing the nm23 gene and have transfected one cell line. Nm23 expression in these transfected cells ranges from 3 to over 50-fold when compared to the non-transfected cells. These cells are presently being analyzed to define affects of over expression of nm23 and relationship to perturbations of proteolytic factors.

Prognostic factors are highly needed to divide node negative breast cancer patients into groups of low versus high risk of recurrence and death. In order to invade and spread, cancer cells must degrade extracellular matrix proteins. Accordingly, tumor levels of molecules involved in this degradation might be associated with patient outcome. Previous work has demonstrated that high levels of the aspartyl protease cathepsin D in breast cancer are associated with a poor prognosis and similar findings have been reported for molecules involved in the urokinase pathway of plasminogen activation. Interactions between different protease systems have been described and data suggest that several proteolytic enzymes may be operable at the same time in a tumor. In the present study we measured cathepsin D (n=162), uPA (n=116), uPAR (n=109) and PAI-1 (n=135) in tumor cytosols obtained from a population of node negative breast cancer patients. A significant correlation was found between levels of uPA, uPAR, and PAI-1. Levels of cathepsin D were directly related to levels of uPA and uPAR. With a median observation time of 4.81 years, univariate survival analyses showed that high levels of uPA and cathepsin D significantly predicted a shorter disease free survival, while only high levels of cathepsin D were able to significantly predict a shorter overall survival. Tumor levels of uPAR and PAI-1 gave mixed results depending on the cut-off point choosen. Interestingly, multivariate analysis demonstrated that PAI-1 and cathepsin D were independent significant prognostic indicators for disease-free survival while only cathepsin D was helpful in overall survival. The five year relapse rate of patients with low PAI-1 and low cathepsin D was 13% while patients who had greater than the median value for both of these molecules had a 5 year relapse rate of 40%. These data would indicate that at least two different protease systems are active in spread of node negative breast cancer and that measurement of these molecules may aid in the decisions to be made when offering adjuvant treatment to these patients.

Retinoids modulate gene activity, cell growth and differentiation by binding to a series of nuclear receptors, i.e., retinoic acid receptors (RARs) or retinoid X receptors. Retinoic acid (RA) inhibition of estrogen receptor (ER)-positive breast carcinoma seems to be mediated through RAR alpha. Estrogens upregulate RAR alpha in ER-positive breast carcinoma cell lines. In this study we examined RAR alpha expression in the ER-positive MCF7 and ER-negative MDA-MB-231 human breast carcinoma cell lines as well as in 10 ER-negative and 9 ER-positive infiltrating ductal breast carcinoma specimens using immunohistochemistry and quantitation by image cytometry. MCF7 cells expressed twofold higher levels of RAR alpha protein than MDA-MB-231 cells. RAR alpha expression, as detected by immunostaining and quantitated by image cytometry, was upregulated in these cells by estradiol. ER-positive breast carcinoma specimens also exhibited approximately two-fold higher RAR alpha levels than their ER-negative counterparts. Thus, RAR alpha expression is significantly elevated in ER-positive breast tumors as assessed by detection and quantitation using immunohistochemical staining and image cytometry, respectively. Whether the decrease in RAR alpha protein levels and loss of RA-mediated growth inhibition in ER-negative tumor plays a role in the increased metastatic potential of ER-negative tumors remains to be determined.

Her2 a key member in the epidermal growth factor receptor (EGFR) family, is one of the most dominant oncogenes in breast cancer. It is overexpressed in approximately 25%-30% of human breast cancers, which confers a more aggressive tumor phenotype and is associated with poor prognosis. The most effective FDA-approved therapy targeting Her2 is the monoclonal antibody trastuzumab (Ttzm; Herceptin). Ttzm inhibits Her2 activation and blocks downregulation of PI3K/Akt activities, which ultimately leads to increased expression of the cyclin-dependent kinase inhibitor p27, cell cycle arrest, and/or apoptosis. However, about 75% of Her2-positive breast tumors are or become resistant to Ttzm and experience relapse or disease progression. This suggests that tumors acquire or possess an intrinsic mechanism of resistance that prevents from Her2 inhibition. Therefore, it is important for the clinic to identify novel pathway to overcome the resistance. Clinicians cannot predict which patients with Her2-positive breast cancer will benefit from this treatment. Despite extensive studies, the mechanisms and genes responsible for resistance to trastuzumab (Ttzm) have not been entirely identified. Thus, novel strategies for treating Her2-positive disease are urgently needed. Recent studies have proposed that resistance to Ttzm is related to IGF-1R overexpression, rapid p27 degradation, PTEN loss, and PI3K mutations, but a clear target for modifying its resistance has yet to be identified. The cytostatic effect of Ttzm correlates with downregulation of Akt activity, which ultimately leads to increased expression of the cyclin-dependent kinase inhibitor p27, cell cycle arrest and/or apoptosis. c-Jun activation domain-binding protein 1 (Jab1/Csn5), a novel candidate oncogene that we identified, contributes to the progression of breast carcinoma and is correlated with poor prognosis. Jab1/Csn5 expression is low or absent in normal adults breast tissues, but over-expressed in 50% of primary breast tumor and 90% of metastatic breast cancer and other tumor types (ovarian cancer, pancreatic, non-small-cell lung carcinoma, and non-Hodgkin's lymphomas). Jab1/Csn5 negatively regulates p27 tumor suppressor gene, mediates p27 nuclear-to-cytoplasmic export and degradation. Jab1 levels are inversely associated with p27 expression in vitro and in vivo, which means that its up-regulation may contribute to trastuzumab (Ttzm) resistance by promoting p27 degradation. This study was conducted to investigate the role of Jab1 in modulating trastuzumab (Ttzm) resistance. We hypothesize that overexpression of Jab1 plays a crucial role in resistance to Ttzm through negative regulation of the cell-cycle inhibitor p27. Using molecular approaches and pre-clinical studies we identified the pathway leading to resistance the Ttzm. In this study, we will test a novel paradigm that inhibition of Jab1 expression renders Ttzm-resistant cells sensitive to Ttzm by stabilizing nuclear p27 expression levels. Finally Jab1 could be used as a predictive marker of tumor response to Ttzm, which could assist clinicians in selecting the best treatment modalities for patients with breast cancer Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B231. Citation Format: Thuy T. Vu, Terry J. Shackleford, Qingxiu Zhang, Francisco J. Esteva, Elias Drakos, Ling Tian, Timothy Kute, Aysegul A. Sahin, George Z. Rassidakis, Francois X. Claret. Jab1/Csn5 a new player driving the resistance to Her2-targeted therapies for breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B231.

As a polycomb group protein, Yin Yang 1 (YY1) plays an important role in cancer epigenetics. YY1 regulates both gene expression and protein modifications, including histone acetylation and methylation. We recently reported a proliferative role of YY1 and its cytoplasmic presence in breast cancer. In the current study, we demonstrated that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. The immunostaining signal of YY1 and AKT(S473) phosphorylation positively correlated and colocalized in breast cancer tissues and cells. The oncogene protein binding (OPB) domain (G201-S226) of YY1 bound to AKT and was necessary for YY1 in promoting AKT phosphorylation. This novel function of YY1 is independent of its transcriptional activity because YY1 promoted AKT phosphorylation in an in vitro setting using purified proteins and YY1 mutants deficient in binding to DNA retained this activity. Our further studies revealed that YY1 interacted with the PH domain of AKT where PIP3 binds and its role in promoting AKT phosphorylation was abolished when we depleted mSIN1, a specific component of mTORC2 that mediates AKT(S473) phosphorylation. Thus, we discovered a PI3K-independent mechanism of mTORC2-mediated AKT activation stimulated by YY1 binding to its PH domain, which supports the proliferative role of YY1 in mammary oncogenesis. Citation Format: Qiang Zhang, Meimei Wan, David A. Horita, Lance D. Miller, Steven J. Kridel, Timothy E. Kute, George Kulik, Guangchao Sui. Yin Yang 1 promotes AKT activation through direct protein binding. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A25.

Membrane-interactive phospholipids (PLs), previously evaluated for activity against HIV-1 in vitro, are known to affect late steps in viral replication. Studies were done to determine the effects of PL analogs on post-translational processing of HIV-1 proteins, binding of viral surface gp160/gp120 to CD4 receptor, and HIV-1-induced cell fusion. Results of this investigation indicated that PL alone (1-octadecanamido-2-ethoxypropyl-rac-3-phosphocholine, CP-51) and PL-AZT conjugate (1-octadecanamido-2-ethoxypropyl-rac-3-phospho-3'- azido-3'-deoxythymidine, CP-92) have no effect on HIV-1-induced syntheses or processing of gp160/gp120, pr51, p24, or p17 (including myristoylation) in infected cells. Progeny HIV-1 particles made in CP-92-treated H9IIIB cells contained gp120, pr51, and p24; however, these virus particles had reduced capacity to bind to CD4+ cells. Both CP-51 and CP-92 inhibited syncytium (cell fusion) formation between treated HIV-1-infected cells and uninfected CD4+ cells, and, they reduced HIV-1 gp160/gp120 binding to CD4+ cells and monoclonal antibody. These results suggest that anti-HIV-1 activity of PL compounds involves alteration of cell surface membranes and viral envelopes. Phospholipid compounds are a novel class of membrane interactive compounds with potential use in blocking the spread of HIV-1 infection and pathogenesis in AIDS.

The mechanism of synergy between 3'-azido-3'-deoxythymidine (AZT) and anticancer agents was investigated with emphasis on cell-cycle events. Exposure of exponentially growing WiDr human colon carcinoma cells to AZT resulted in synchronization of cells in the S phase of the cell cycle. Following treatment with AZT at 50 or 200 microM, 62% +/- 3% or 82% +/- 4% of the cells were in the S phase as compared with 36% +/- 2% in the control. Bromodeoxyuridine uptake studies revealed that the synchronized cells actively synthesized DNA. At concentrations of up to 200 microM, AZT produced a cytostatic rather than cytotoxic effect as indicated by viability and cell growth measurements. At 200 microM, AZT-induced synchronization was significant (P =0.001) after 12 h of drug exposure, reached a maximum at 24 h, and reversed to baseline levels by 72 h even in the continued presence of the drug. This indicates that AZT-induced cytostasis is a transient and reversible effect. The cell-cycle events seen with AZT in WiDr cells were also observed in eight of nine human tumor cell lines tested. Isobologram analysis of WiDr cells preexposed to AZT for 24 h and then exposed to either AZT-5-fluorouracil or AZT-methotrexate for a further 72 h revealed synergy between AZT and the anticancer agents, indicating that AZT-induced synchronization may have therapeutic benefits.

Her2-positive (Her2+) breast cancer has the second worst prognosis of the five subtypes of breast cancer. The most successful targeted therapy for Her2+ breast cancer to date is trastuzumab (Herceptin)-based. However, the median duration of response to trastuzumab is shorter than 1 year, and about 75% of patients who initially responsed, has experienced resistance to the therapy after a year. Thus, better treatment strategies for Her2+ subtype are urgently needed. We and other researchers previously demonstrated that c-Jun activation domain-binding protein 1 (Jab1) negatively regulates p27, mediates p27 nuclear-to-cytoplasmic export and degradation and contributes to the loss of p27 that is seen in >50% of breast tumors and that correlates with poor clinical outcome in breast cancer cells. Thus, implicates the potential role of Jab1 in interfering with trastuzumab. We identified Jab1/Csn5 as a novel oncogene. Jab1 is amplified and overexpressed in elevated in 50% of primary and 90% of metastatic breast tumors, but is low or absent in normal adult breast tissue. In addition, high Jab1 expression is associated with short progression-free survival durations in breast cancer patients. Our preliminary data showed that inactivation of Jab1/Csn5 in trastuzumab-resistant breast cancer cells sensitized the cells to trastuzumab in a dose- and time-dependent manners. However, the ways in which Jab1 expression is up-regulated in Her2+ breast cancer cells and how this up-regulation drives trastuzumab resistance remain largely unclear. In this study, we will seek to analyze the cross-talk between Src/Stat3 or Akt signaling and Jab1 activation in breast carcinoma. We have found that Stat3 is a novel positive regulator of Jab1 expression in breast cancer cells and that Jab1 overexpression driven by the Src/Stat3 pathway compensates for trastuzumab's inhibition of Her2 signaling in Her2+ breast cancer cells. This suggests that activated Stat3, by up-regulating Jab1 expression, inhibits trastuzumab-induced cell-cycle arrest. In general, our proposed study elucidates Jab1 as a novel contributor to trastuzumab resistance and determine its potential as a prognostic and predictive marker as well as an important therapeutic target. The successful completion of our study would benefit breast cancer patients in three ways. 1) Because Jab1 is rarely expressed in mammary epithelial cells, targeting Jab1 would be less toxic to normal epithelial cells, compared to other agents. 2) Combining Jab1 with trastuzumab would interfere with both Src/Stat3 and Akt pathway. This would significantly benefit patients with Her2+ breast cancer refractory to this therapy due to PTEN loss or activating mutant Stat3. 3) Because Jab1 expression is higher in trastuzumab-resistant cells than in trastuzumab-sensitive cells, Jab1 could be used as a marker of tumor response to trastuzumab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1912. doi:1538-7445.AM2012-1912

Background: The killing of Her2 + breast cancer cells by the antibody trastuzumab is an attractive therapy because of its specificity and limited side effects. However, many Her2 + positive patients have innate resistants or will become resistant to this treatment. Although Her2 status can be determined by immunohisochemistry or FISH, still only 30% of patients positive for high Her2 status will respond to trastuzumab alone. Improved methods to predict sensitivity to trastuzumab are greatly needed. Since trastuzumab is believed to work by antibody-dependent cell-mediated cytotoxicity (ADCC), we have developed a novel and reproducible bioassay to measure trastuzumab-dependent ADCC which may be predictive of patient response. Methods: Using both an xCELLigence platform (Roche Applied Science) and a flow cytometry assay (PanToXiLux from OncoImmunin, Inc), we measured the ability of mononuclear cells (MNCs) to kill four different breast cancer cell lines in the presence or absence of trastuzumab. Two cell lines used have resistance to trastuzumab and two cell lines have lower Her-2 expression. The xCELLigence platform measures killing in real time while the flow cytometry assay measures the transfer of granzyme B and upstream caspase activity from mononuclear cells to the tumor cells and the activation of cell death processes. The ability of mononuclear cells to bind tumor cells in the presence and absence of trastuzumab was evaluated by microscopy. Using miltenyi beads and immunofluroescence, we have measured which MNC population is most effective in ADCC activity. MNCs from patients with breast cancer and from individuals without disease were analyzed. Results: These studies provide a simple and reproducible assay for ADCC activity. Effective ADCC killing is directly related to Her2 expression on the tumor target. The degree of killing is directly related to the number of mononuclear cells added in the study when trastuzumab is added. Addition of trastuzumab alone to these cell lines results in little killing. MNCs from non-diseased individuals as well as those obtained from breast cancer patients display a broad range of ADCC killing efficiency per cell. Finally, our results demonstrate that NK cells are the major MNC population of effect killing. Conclusion: The two assays presented here and other specific studies show that trastuzumab can work with the immune system to increase cell killing of Her2 overexpressing cells by ADCC. A correlation between the results of these assays and the clinical response to trastuzumab will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5374. doi:1538-7445.AM2012-5374

Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008-32 microM bromodeoxyuridine (Brd-Urd). After dual labeling with monoclonal anti-BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8-32 microM--a 40-fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady-state plasma concentrations in the 8.0 microM range can be expected to provide a reliable estimate of the S-phase fraction.

Estrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence-E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension. Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein-labeled estrogen in fluorescein-E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 × 108 M−1 for the ER and is a single component as determined by HPLC. Specific fluroescent uptake of coumestrol was performed on ER+ and ER− viable cell suspensions. When these coumestrol-cell suspensions were excited at 350–360 nm and the blue emission was measured using flow cytometry, the result was a fluroescence uptake that was not highly displaceable by exess nonfluorescence E2 probes. Further studies on the modification of several parameters of the incubation step to enhance specific fluorescent uptake were also unsuccessful. Coumestrol was, however, a good competitor for both cellular and nuclear uptake of 3H-E2.

The prime importance of axillary node status in predicting recurrence and survival has been appreciated for a long time. More recently routine measurements of estrogen and progesterone receptors have added to our prognostic abilities. The next generation of prognostic markers to be used in the routine clinical setting will be measures of tumor aggressiveness which will prompt therapeutic decisions. The technique of flow cytometry can provide clinicians with two new important pieces of information. First, it gives a measurement of the percentage of cells in S phase of the DNA replicative cycle, in other words, how fast a tumor is growing. Second, it assesses the aneuploidy, or total amount of extra DNA in the tumor cell, which appears to correlate with malignant aggressive tumor behavior.

The cell cycle kinetics of normal mammary tissue and cancer mammary tissues were studied by flow cytometry to determine the effect of estrogen and progesterone and antiestrogens on proliferative activity. The proliferative activity was higher in rat mammary tissue during the estrus and pregnant stages compared to the nonestrus or lactational stages. Upon 5 days administration of estrogen and progesterone to the lactating animal, the low proliferative activity was increased to the values found in the pregnant stage. Steroidal effect on proliferative activity was also shown using organ cultures of normal rat tissues and the increase in proliferative activity can be reversed by the addition of 10(-6) M retinoic acid but not by retinyl acetate. In a human mammary tumor cell line (MCF-7), the cell kinetic activity was significantly depressed by antiestrogens (i.e., tamoxifen) as determined by both flow cytometry and tritiated thymidine uptake studies. Finally, a human tumor model system was developed with the MCF-7 cell line in estrogenized nude mice. Flow cytometry of the resulting tumor indicates a tetraploid tumor similar to the cell culture. Cell kinetic analysis of these nude mice tumor cells demonstrates the high proliferative activity of the estrogenized animals compared to the tamoxifen-treated animals. In conclusion, the use of flow cytometry to rapidly measure cell kinetic activity of mammary tissue has been demonstrated. The effects of estrogen or estrogen and progesterone hormones seem to increase the proliferative activity, while antiestrogens decrease this activity. Findings are observed in both in vivo and in vitro conditions or normal tissues, mammary cancer tissue, and cultured cells.

This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.

Improvements on the steroid receptor assay have been developed which eliminate tedious procedures and give faster and more accurate results. A protein assay (Waddell method) is discussed which directly measures peptide bonds in cytosolic material. It is more rapid and more sensitive than the Lowry method. Programs for the analysis of data obtained by either the dextran-coated charcoal method or the sucrose density gradient have been written. These are superior to previously published programs and can be incorporated into any laboratory with a minimum of cost. The program for the dextran-coated charcoal method converts the counts per minute from both total and nonspecific 3 H-steroid binding activity into information needed for a Scatchard analysis. These data are plotted and a linear regression and determination coefficient for best fit of the data are performed. In the sucrose density gradient method program, the counts per minute from both total and nonspecific 3 H-steroid binding activity is converted to disintegrations per minute and plotted versus the corresponding fraction number. The specific binding activity in femtomoles/milligram of protein in the various molecular forms can then be obtained through set programmed steps.

The potential significant therapeutic and prognostic roles for the sex steroid receptors in ovarian cancer are recognized. The authors present in detail the biochemical, morphologic, cytogenetic, and growth characteristics of an ovarian carcinoma cell line, BG-1, which has functional estrogen and progesterone receptors (23 and 300 fmol/mg protein, respectively) in clinically significant levels. In particular, BG-1 has a DNA index of 1.14, a stable karyotype with specific translocations, and produces and secretes CA 125 into the media.

Meningiomas have a wide range of biological potential and clinical behavior. Histological findings are helpful in recognizing the malignant potential of a given tumor, but often fail to correlate with gross features, liability of recurrence, and extent of associated cerebral edema. To find alternate approaches to improve the correlation between biological and clinical behavior, 20 meningiomas were studied by flow cytometry (FC), an assessment that has been applied to meningiomas previously. Such FC features as DNA index (DI) and proliferative index (PI, %G2 † %S) were correlated with size, location, brain invasion, associated edema, and recurrence. Tumors with severe edema had significantly higher PIs (19.5 ± 4.1) than those with moderate (12.6 ± 4.5) or minimal (8 ± 0) edema (P < 0.05). The PI was greater than 16 in those tumors that recurred (n = 3) or invaded the brain (n = 3). Six tumors were aneuploid (DI, 1.33 ± 0.17; PI, 17.1 ± 5.3). These were uniformly large when compared with the diploid tumors, which were more variable in size. All of the aneuploid tumors were associated with moderate to severe cerebral edema. Two partly psammomatous tumors with high PIs and foci of high cellularity suggesting recent growth were associated with severe edema. One of these exhibited brain invasion. These preliminary data indicate that FC may have a potential use in the clinical management of meningiomas.

With endometrial adenocarcinoma, several factors, such as stage and histologic grade, are well recognized as having prognostic significance. Other variables, e.g., nuclear grade, probably are also important clinically. A preliminary investigation of the prognostic value of the ploidy, cell cycle kinetics, and sex steroid receptor contents of these tumors has been conducted and compared with more conventional prognostic factors such as stage, grades, and depth of invasion. As analyzed by flow cytometry, the proliferative activities of the carcinomas were significantly associated with prognosis. Tumors with lower levels of proliferative activity were related to improved survivals. Two thirds of the carcinomas were diploid; ploidy did not carry prognostic weight in the series. Both the estrogen and progesterone receptor status were related to survival. Patients whose tumors were positive for these receptors had a better prognosis than did those in which the receptors were not present in significant quantities.

Yin Yang 1 (YY1) is highly expressed in various types of cancers and regulates tumorigenesis through multiple pathways. In the present study, we evaluated YY1 expression levels in breast cancer cell lines, a breast cancer TMA, and two gene arrays. We observed that, compared with normal samples, YY1 is generally overexpressed in breast cancer cells and tissues. In functional studies, depletion of YY1 inhibited the clonogenicity, migration, invasion, and tumor formation of breast cancer cells, but did not affect the clonogenicity of nontumorigenic cells. Conversely, ectopically expressed YY1 enhanced the migration and invasion of nontumorigenic MCF-10A breast cells. In both a monolayer culture condition and a three-dimensional Matrigel system, silenced YY1 expression changed the architecture of breast cancer MCF-7 cells to that resembling MCF-10A cells, whereas ectopically expressed YY1 in MCF-10A cells had the opposite effect. Furthermore, we detected an inverse correlation between YY1 and p27 expression in both breast cancer cells and xenograft tumors with manipulated YY1 expression. Counteracting the changes in p27 expression attenuated the effects of YY1 alterations on these cells. In addition, YY1 promoted p27 ubiquitination and physically interacted with p27. In conclusion, our data suggest that YY1 is an oncogene and identify p27 as a new target of YY1.