bluuebunny/crossref_metadata_2025 huggingface dataset:
First-Line Bevacizumab (Bev) Combination Therapy in Triple-Negative (TN) Locally Recurrent/Metastatic Breast Cancer (LR/MBC): Subpopulation Analysis of Study MO19391 in >2000 Patients (Pts).
Authors: Thomssen C. , Pierga J. , Pritchard K. , Biganzoli L. , Cortes-Funes H. , Petrakova K. , Kaufman B. , Smith I.
Published: December 2010

Background: The combination of Bev with taxane-based therapy, anthracycline-based combination therapy, or capecitabine significantly improves progression-free survival versus chemotherapy alone in LR/MBC, as shown in three randomized, phase III trials (E2100, AVADO, RIBBON-1). Pts with TN LR/MBC appeared to derive similar benefit from Bev compared with non-TN pts in subanalyses of the E2100 and AVADO trials. We conducted a subpopulation analysis of TN pts treated with Bev combination therapy in the MO19391 observational study, which included a broader pt population reflecting general oncology practice.Methods: Pts with HER2-negative (or trastuzumab-pretreated HER2-positive) LR/MBC received first-line Bev 10 mg/kg q2w or 15 mg/kg q3w in combination with taxane-based therapy or other non-anthracycline-containing regimen, according to physician preference. Bev was continued until disease progression. The primary endpoint of MO19391 was safety. Secondary endpoints included time to progression (TTP) and overall survival.Results: 484 of the 2041 pts in MO19391 had TN LR/MBC and 1346 had non-TN disease. The remaining 211 had missing or unknown ER, PgR, or HER2 status and were excluded from this analysis. The proportion of pts with disease-free interval (DFI) ≤24 months was considerably higher in the TN population than in non-TN pts. As expected, the safety profile of Bev combination therapy showed no differences between the two subpopulations. The only pre-defined Bev adverse events of special interest reported at grade ≥3 in >1% of TN pts were hypertension (4.5%) and pulmonary embolism (1.4%). Baseline characteristics, treatment exposure, and efficacy are summarized below. Overall survival data are immature. TN (n=484)Non-TN (n=1346)Median age, years (range)52 (24–85)54 (21–93)DFI ≤24 months, %5525>3 metastatic lesions, %6470Mean duration of Bev, months (range)5.6 (0.0–21.0)7.6 (0.0–25.6)TTP*  Events, n (%)297 (61)656 (50)Median, months (95% CI)7.1 (6.4–7.6)10.6 (10.0–11.1)Response rate, %*48.354.5Complete response9.66.9Partial response38.847.6*n=483 in the TN group; n=1322 in the non-TN groupConclusions: In this ongoing study, Bev combination therapy demonstrated a 48% response rate and median TTP of 7.1 months in pts with TN LR/MBC. Findings from this observational study are consistent with retrospective analyses of randomized phase III trials. Prospective trials evaluating Bev combination regimens in TN breast cancer (adjuvant and metastatic settings) are ongoing.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6093.
A Phase (Ph) I/II Study of CR011-VcMMAE, an Antibody-Drug Conjugate, in Patients (Pts) with Locally Advanced or Metastatic Breast Cancer (MBC).
Authors: Burris H. , Burris H. , Saleh M. , Bendell J. , Bendell J. , Hart L. , Hart L. , Rose A. , Dong Z. , Siegel P. , Crane M. , Donovan D. , Crowley E. , Simantov R. , Vahdat L.
Published: December 2010

Background: Glycoprotein NMB (GPNMB), also known as osteoactivin, is expressed in 25-40% of breast cancers and is associated with an increased risk of recurrence. This is the first study of CR011-vcMMAE, a fully-human monoclonal anti-GPNMB antibody conjugated to the tubulin inhibitor monomethylauristatin E (MMAE), in pts with breast cancer.Methods: Eligible pts with MBC had ³ 2 prior chemotherapy regimens, including a taxane, an anthracycline, and capecitabine; and ECOG PS ≤ 2. Doses were escalated to 1.88 mg/kg IV q3w (the maximum tolerated dose [MTD] in a previous study of patients with melanoma) using a standard 3+3 design followed by a Ph II expansion at the MTD. Pts received CR011-vcMMAE until disease progression (PD) or intolerable toxicity. The primary endpoint of the Ph II study was 12-week progression free rate, defined as the proportion of patients who were alive and free of progressive disease 12 weeks from the first dose of CR011-vcMMAE. A Simon two-stage minimax design was used (p0=0.1; p1=0.3, α=β=0.1) with 16 patients in the first stage and a total of 25 pts. Secondary endpoints included objective response rate and duration of response. IHC with a goat polyclonal antibody to GPNMB was performed on pt biopsy specimens.Results: In Ph I, 14 pts were treated with CR011-vcMMAE at 1.0 mg/kg (n=3), 1.34 mg/kg (n=5), and 1.88 mg/kg (n=6). In the first 2 pts at 1.34 mg/kg, dose limiting toxicity of worsening peripheral sensory neuropathy was observed. Pts with baseline neuropathy worse than grade 1 were subsequently excluded. There were 3 partial responses, one confirmed, and 1.88 mg/kg q3w was selected for Ph II. In Ph II, 14 pts with MBC (median age 57 years, range 34 - 76) had a median of 5 prior regimens; median follow up was 6 weeks; 12 pts are ongoing (median duration 6 weeks, range 1 - 14 weeks). Two pts were progression-free at 12 weeks, therefore the study has met the criteria for advancement to the second stage. The most common AEs were rash (61%), fatigue (50%) and alopecia (50%). The most common grade 3/4 AEs were neutropenia (17%) and neuropathy (11%). IHC staining of 5 patient biopsies revealed 2 with positive GPNMB expression, including one of the patients with PR.Conclusions: CR011-vcMMAE is active and well-tolerated in heavily pretreated pts with advanced breast cancer. The Phase II study has met the criteria for advancement into the second stage, and enrollment is ongoing.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6096.
Updated Survival Analysis of a Randomized Study of Lapatinib Alone or in Combination with Trastuzumab in Women with HER2-Positive Metastatic Breast Cancer Progressing on Trastuzumab Therapy.
Authors: Blackwell K. , Burstein H. , Sledge G. , Stein S. , Ellis C. , Casey M. , Baselga J. , O'Shaughnessy J.
Published: December 2010

Background: The synergistic interaction of lapatinib combined with trastuzumab was established in HER2-positive preclinical models, hence providing the rationale to evaluate this combination in a clinical setting. Progression-free survival (PFS) from study EGF104900 revealed the combination of lapatinib plus trastuzumab was superior to lapatinib alone in women with HER2-positive metastatic breast cancer (MBC) that progressed on multiple lines of trastuzumab-based therapy. Preliminary data showed a trend in overall survival (OS) favoring the combination therapy; however, data were not mature. Updated OS analyses are reported.Methods: Women with HER2-positive MBC progressing on prior trastuzumab-containing regimens were randomized to receive either lapatinib 1500 mg once daily or lapatinib 1000 mg once daily in combination with trastuzumab 2 mg/kg (after a 4-mg/kg loading dose). If objective disease progression occurred on or after 4 weeks of lapatinib alone, crossover to the combination arm was permitted. OS was summarized using Kaplan-Meier curves and compared between treatment arms using stratified log-rank tests. Analyses adjusting for baseline prognostic factors and crossover were also performed.Results: 296 women were randomized (148 per arm). The median number of prior trastuzumab-containing regimens for MBC treatment was 3. Of the women randomized to lapatinib alone, 52% (77/148) crossed over to the combination arm. At data cut-off for updated OS, 218 deaths (74%) had occurred. Median OS following treatment with lapatinib plus trastuzumab was 60.7 weeks compared with 41.4 weeks for lapatinib alone. A significant improvement in OS was demonstrated with combination therapy compared with lapatinib monotherapy (HR: 0.74; 95% CI: 0.57, 0.97; P=.026). The survival benefit was maintained after adjusting for baseline prognostic factors (HR: 0.71; 95% CI: 0.54, 0.93; P=.012). A trend toward a clinically relevant 25% reduction in risk of death (P=.080) was also observed after adjusting for crossover.Conclusion: A statistically significant OS benefit was observed in women with heavily pretreated, HER2-positive MBC treated with lapatinib in combination with trastuzumab compared with those treated with lapatinib alone. The actual survival benefit of the combination therapy may be underestimated due to the high frequency of crossover.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 61.
Combination Regimens of nab-Rapamycin (ABI-009) Effective Against MDA-MB-231 Breast-Tumor Xenografts.
Authors: Desai N. , D'Cruz O. , Trieu V.
Published: December 2010

Background: The mammalian target of Rapamycin (mTOR) is a key intracellular kinase integrating cell proliferation and survival and is an attractive target for cancer therapy. Nab-rapamycin (ABI-009) was developed using proprietary nab-technology and showed dose-linear pharmacokinetics, safety up to 90 mg/kg and effective antitumor activity at 40 mg/kg in the human-tumor-xenograft panel. The goal of this study was to test the effectiveness of different combination regimens for ABI-009 against MDA-MB-231 breast-tumor xenografts.Materials and methods: Subcutaneous human-breast (MDA-MB-231) tumors were grown in athymic nude mice and treated intravenously (IV) with ABI-009 alone at 40 mg/kg (3xwkly/4wks) and in combination with Erlotinib (50 and 100 mg/kg/day/4wks, IP), Cetuximab (20 and 40 mg/kg, 3xwkly/4wks, IP), Doxorubicin (2 and 5 mg/kg, wkly/10 wks, IV), Oxaliplatin (5 and 10 mg/kg wkly/4 wks, IV), SAHA (50 mg/kg, qdx7 or qdx14, IP), and Perifosine (30 and 60 mg/kg, 3xwkly/4wks, PO). The animal weights and tumor measurements were recorded three times weekly and adverse observations recorded.Results: ABI-009 was highly effective as a single agent against MDA-MB-231 breast tumor xenografts with 75% tumor-growth inhibition (TGI). Combination with Erlotinib resulted in TGI of 85% and 95% (50 and 100 mg/kg, respectively), 83% and 87% for Cetuximab (20 and 40 mg/kg, respectively), 83% and 90% for Doxorubicin (2.5 and 5 mg/kg, respectively), 82% and 85% for Oxaliplatin (5 and 10 mg/kg, respectively), 82% and 90% for SAHA (7 and 14 day treatment, respectively), and 92% and 96% for Perifosine (30 and 60 mg/kg, respectively). Notably, Erlotinib and Perifosine were found to synergize with ABI-009 resulting in maximum TGI and tumor growth delay.Discussion: ABI-009 (nab-rapamycin) alone was highly effective against MDA-MB-231 human-breast-tumor xenografts. Antitumor activity of ABI-009 was significantly increased in combination with kinase inhibitors, Erlotinib (EGFR-kinase inhibitor) and Perifosine (AKT inhibitor). Antitumor activity of ABI-009 was also significantly increased in combination with Doxorubicin – a topoisomerase inhibitor, and SAHA – an HDAC inhibitor, but not in combination with Oxaliplatin – a DNA crosslinker. In contrast, combination of ABI-009 with anti-EGFR monoclonal antibody, Cetuximab was not effective. The synergy of these combinations confirmed that rapamycin is active only on TORC1 and that suppression of TORC2 via AKT or PI3K pathways is a means of increasing activity of mTOR inhibitors with potential relevance to breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6106.
Triple-Negative Breast Cancers Express Receptors for GnRH and Can Be Effectively Targeted by the Orally Active GnRH-Antagonist AEZS 115.
Authors: Engel J. , Schreiber A. , Seitz S. , Köster F. , Gaiser T. , Kämmerer U. , Dietl J. , Honig A.
Published: December 2010

IntroductionAEZS-115* is an orally active peptidomimetic antagonist of GnRH, which has been shown to block the pituitary GnRH receptor. In various tumors an autocrine growth promoting loop consisting of GnRH secreted by the malignant cells and tumoral GnRH receptors has been described. The current study investigates the expression of GnRH-receptors in tumor samples of triple-negative breast cancers and evaluates the anti-tumor activity of the GnRH antagonist AEZS 115 in in vitro models of triple negative breast cancers.Study design17 human samples of triple negative breast cancers were tested for GnRH-receptor expression by immunohistochemistry. Human triple negative breast cancer cell lines MDA-MB 468, HCC 1806 and HCC 1937 were analyzed for GnRH receptor expression by immunocytochemistry and western blot analysis. These cell lines were incubated with increasing concentrations of AEZS-115, peptidic GnRH-antagonist Cetrorelix and GnRH-agonist Triptorelin (1, 10, 100 µm) for 48 hours and the number of viable cells was determined by crystal violet staining as well as by ATP-dependent luminometric assay.ResultsPositive staining for GnRH-receptors was detected in all tumor samples (2 high, 12 moderate, 3 weak). Cell lines MDA MB 468, HCC 1806 and HCC 1937 expressed GnRH receptors as demonstrated by immunocytochemistry and western blot analysis. GnRH-antagonist AEZS 115 dose dependently inhibited growth of all three cell lines, while GnRH-agonist Triptorelin and peptidic GnRH antagonist Cetrorelix showed marginal growth inhibition. At 10 µm AEZS-115 inhibited cell growth by 40-60%, at 100 µm growth inhibition was 60-80%. These results obtained by crystal violet staining were confirmed by additional luminometric evaluation of the ATP content after treatment with the respective substances.ConclusionsThe orally active, peptdidomimetic GnRH antagonist AEZS-115 showed substantial anti-tumor activity in these cell lines, while GnRH-agonist Triptorelin and peptidic antagonist Cetrorelix showed only minor growth inhibition. Thus, in triple negative breast cancers the peptidomimetic antagonist AEZS-115 was more potent than the decapeptide Cetrorelix. This finding could be explained by atypical GnRH-receptors on tumor tissue to which the different GnRH-antagonists might bind with different affinity. Due to high receptor expression the GnRH receptor is a promising target in triple negative breast cancers. The good antitumor-activity makes AEZS-115 a promising candidate for in vivo studies in this tumor entity.*Aeterna Zentaris GmbH, Frankfurt/M, Germany.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6108.
A Type I Insulin-Like Growth Factor Receptor Kinase Inhibitor (PQIP) Enhances the Cytotoxicity of Doxorubicin in Human Cancer Cell Lines.
Authors: Zeng X. , Buck E. , Yee D.
Published: December 2010

The type-I insulin like growth factor (IGF-IR) is either activated and/or overexpressed in a wide range of tumor types and contributes to tumorigenicity, proliferation, survival, metastasis and drug resistance. Disruption of IGF-IR signaling alone or in combination with other cytotoxic agents has emerged as an important strategy in cancer therapy. Our laboratory has shown that sequentially combining doxorubicin with anti-IGF-IR antibodies significantly enhances the response of chemotherapy, while the opposite sequence causes cells resistant to chemotherapy. Here, we show that a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP) inhibited IGF-IR and insulin receptor (IR) kinase activity and proliferation in MCF-7 and MDA435/LCC6 cell lines. Moreover, PQIP treatment blocked both IGF-I and insulin stimulated activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase in both cell lines. At doses that inhibited monolayer cell growth, the compound also inhibited cell motility, blocked IGF-I stimulated S-phase progression and induced autophage; at higher doses, it also caused PARP cleavage. Combining PQIP with doxorubicin significantly enhanced cytotoxicity but did not further enhance doxorubicin-induced PARP cleavage in monolayer cell growth. Furthermore, our sequencing studies showed that combining PQIP with doxorubicin simultaneously or doxorubicin followed by PQIP significantly inhibited the anchorage-independent growth in both MCF-7 and MDA435/LCC6 cells. In contrast, pre-treatment with PQIP followed by doxorubicin did not enhance the cytotoxicity of doxorubicin in anchorage-independent growth, which is similar as anti-IGF-IR antibodies in combination with doxorubicin. In summary, these results suggest that the IGF-IR tyrosine kinase inhibitor PQIP can be used alone or in combination with chemotherapy to enhance cytotoxicity of human tumor cell lines, and the timing of IGF-IR inhibition may affect responses to doxorubicin.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6110.
Delivery of Therapeutic Nanoshells to Hypoxic Areas of Tumors Using a Cellular Trojan Horse.
Authors: Choi M. , Bardhan R. , Badve S. , Nakshatri H. , Halas N. , Clare S.
Published: December 2010

Background: The goal of this project is to develop a novel and innovative delivery system for anti-tumor therapeutics which circumvents the impediments to efficient delivery that are present in a solid malignancy. Malformed blood vessels, elevated interstitial pressure, and large transport distances in a tumor, individually or in concert limit or thwart the diffusion of oxygen, glucose and soluble therapeutics. This results in hypoxia, necrosis, and makes regions of the tumor inaccessible to otherwise efficacious therapies. In addition, hypoxia exerts a selective pressure on tumor cells in that only those with an aggressive phenotype are able to survive hypoxia; these cells may be the source of subsequent local recurrence and distant metastasis.Monocytes/macrophages respond to intratumoral hypoxia and necrosis and are recruited by a chemoattractant gradient. Our strategy is to take advantage of the recruitment of monocytes/macrophages by a tumor, and of the monocytes/macrophages innate phagocytosis ability in order to load them with nanoshells, and thereby to create a Trojan horse. This study was designed to determine if monocytes/macrophage loaded with gold-silica nanoshells would respond to the chemoattractant gradient and become tumor associated macrophages. Nanoshells can convert absorbed light into thermal energy, which can be utilized for photothermal ablation. Therefore, our xenograft model system was utilized also to test whether malignant epithelial cells can be successfully be treated by thermal ablation of neighboring macrophages.Materials and Methods: A human breast tumor xenograft, and a separate metastatic brain tumor xenograft were established in nude mice. Monocytes were isolated from human whole blood and loaded with nanoshells. The nanoshells were conjugated with a near-infrared dye. The loaded macrophages were injected via the tail vein into the systemic circulation of the mice. The tumors were imaged by fluorescence imaging and magnetic resonance imaging before and after irradiation with a near-infrared laser (0.5 W, 5mm spot size) for varying lengths of time.Results: The in vivo delivery of the macrophages loaded with nanoshells to the human xenograft tumors was verified. Significant uptake of loaded macrophages in tumor was observed at 24 hours after injection. Uptake was also observed in the lungs, liver and spleen. An increase of ∼ 30°C at the tumor skin was noted following 90 seconds of irradiation of the orthotopic tumor.Discussion: Nanoshell-loaded macrophages respond to the presence of an orthotopic breast tumor and to breast cells metastatic to the brain, exit the circulation and accumulate in the rim of hypoxic cells surrounding necrosis. Photothermal ablation of the tumor cells infiltrated by this innovative therapeutic delivery method produces a targeted therapy in that it targets the tumor hypoxia.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6113.
The Ribonucleoside Analog, 8-Chloro-Adenosine, Inhibits the mTOR Pathway and Induces Autophagy.
Authors: Stellrecht C. , Shentu S. , Gandhi V. , Gandhi V.
Published: December 2010

In contrast to deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-chloro-adenosine (8-Cl-Ado) possesses a ribose sugar. This unique nucleoside analog is RNA directed and is in a phase I clinical trial for hematological malignancies. Preclinically, it has been also found to be cytotoxic to a number of epithelial and neuronal cell lines. Previously, we demonstrated that in the breast cancer cell lines, MCF-7 and BT-474, 8-Cl-Ado accumulates as a triphosphate reaching its plateau by 6 h of treatment with 10 μM 8-Cl-Ado. This accumulation was coupled with a parallel decrease in the level of the endogenous ATP pool by 60 and 40%. These changes in nucleoside levels then lead to ∼50% inhibition of RNA synthesis. The net effect of these metabolic changes results in a growth inhibition of ∼90% after 5 d treatment with 10 μM 8-Cl-Ado as measured by MTS assay. This growth inhibition was not due to a cell cycle arrest. Additionally, flow cytometry analysis of annexin V staining showed only a modest induction of apoptosis first detected within 24 h of treatment and reached ∼30% by 5 d. Because the low level of apoptosis can not account for high percent of growth inhibition suggests alternative mechanism(s) for the cytotoxic activity of 8-Cl-Ado. Depletion of the cellular ATP pool is often associated with the activation of AMP-activated protein kinase (AMPK) through its phosphorylation on Thr172. Correspondingly, we demonstrated that 8-Cl-Ado treatment of MCF-7 and BT-474 cells induced AMPK Thr172 phosphorylation within 7 h. Once activated, AMPK alters various energy regulating signaling cascades which includes the mTOR pathway. Inhibition of mTOR leads to a decrease of energy depletion by reducing protein translation and promotes nutrient restoration through the induction of autophagy. 8-Cl-Ado treatment of MCF-7 and BT-474 cells was associated with a decrease in the phosphorylation of the mTOR translation targets, 4E-BP1 and p70S6K within 8 h of treatment. Likewise, there was an increase in the conversion of microtubule-associated protein 1 light chain 3B (LC3B)-I to its lipidated form, LC3B-II, which is an autophagosome marker. Consistent with the immunoblot analysis, fluorescence microscopic analysis of 8-Cl-Ado-treated MCF-7 cells and BT-474 cells expressing a GFP-LC3 construct also demonstrated an increase in the formation of autophagosomes. Finally, acridine orange staining of 8-Cl-Ado treated MCF-7 and BT-474 cells demonstrated the formation autophagolysosomes. This was further confirmed in MCF-7 cells by monodansylcadaverine staining. In conclusion, our data indicates that 8-Cl-Ado treatment induces autophagy in breast cancer cells which in part elicits its growth inhibitory effects on these cells. Based on this biological activity, we are planning to test 8-Cl-Ado in the clinic for patients with breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6116.
Proteomic Discrimination of Well- from Poorly-Differentiated Breast Carcinomas.
Authors: Ellsworth R. , Seeley E. , Ellsworth D. , Deyarmin B. , Hooke J. , Sanders M. , Caprioli R. , Shriver C.
Published: December 2010

Background: Pathological grade is a useful prognostic factor for stratifying breast cancer patients into favorable (well-differentiated tumors) and less favorable (poorly-differentiated tumors) outcome groups. The current system of tumor grading, however, is highly subjective and a large proportion of tumors are characterized as intermediate-grade, making determination of optimal treatments difficult.Methods: Primary breast tumor specimens from patients diagnosed with well- (n=27) and poorly-differentiated (n=51) invasive ductal carcinoma were obtained from patients enrolled in the Clinical Breast Care Project. Frozen tissues were sectioned and mounted on gold coated MALDI target plates for protein expression profiling. Hematoxylin and eosin (H&E) stained slides were prepared from serial sections for histological characterization. MALDI matrix was deposited as individual spots on the tissue sections in a histology directed manner to assay specific areas and tissue types of interest. Mass spectral data were then acquired from multiple sites across each tissue section.Results: 129 features were observed in well-differentiated and 132 in poorly-differentiated tumors. While the majority of features detected were similar between the two groups, 6 protein features were expressed at significantly lower and 12 at significantly higher levels in the poorly-differentiated tumors, including increased expression of Calgranulin A and Calgizzarin.Conclusions: Protein expression differences detected here suggest that well- and poorly-differentiated invasive breast tumors are molecularly distinct diseases and that these protein changes may contribute to the structural integrity of the tumor cell. In particular, calgranulin A and calgizzarin are members of the S100 protein family, and function in processes such as cell proliferation and differentiation. Further refinement of this differentiation protein signature may not only improve our understanding of the biological processes involved with tumor grade but provide pathologists with new molecular tools to classify breast tumors and reduce the subjectivity associated with current grading criteria.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6126.
The Role of Stromal Cells in Tumor Metastases.
Authors: Pieters A. , Duda D. , Steller E. , Fukumura D. , Jain R.
Published: December 2010

Background: In 1889, Stephen Paget hypothesized that metastatic cells - the “seeds” - only grow in the secondary sites - the “soil” - with a permissive microenvironment. Here, we show that the “seeds” can carry their own “soil” - activated fibroblasts from primary site of metastatic tumors. Moreover, we report that carryover of these non-malignant host-derived cells during metastatic cell colonization in the lungs can increase cancer's metastatic efficiency.Materials and Methods: We use multiple animal models and two metastatic tumor cell lines (LLC1 and LA-P0297) to study the composition, origin and role of fragments shed by tumors in the metastatic process.Results: we have shown that, in addition to single cells (81%), tumors shed fragments consisting of both tumor and host cells. We show that these fragments are more viable in the circulation compared to tumor cells alone (p<0.05). When we selectively deplete the host cells in the tumor microenvironment the spontaneous formation of metastases is significantly reduced three weeks after primary tumor resection compared to control mice (p<0.05). Next, survival was significantly increased in mice with metastases after co-implantation of carcinoma-associated fibroblasts (CAFs) isolated from breast cancer patients together with LLC1 cells, but had no effect in mice implanted with LLC1 cells alone (p<0.01) Lastly, we have assessed the nature of these host stromal cells using immunohistochemistry and RT-PCR and indentified the majority of them as being of mesenchymal/fibroblast lineage.Discussion: We found that tumor-associated stromal cells can escape primary tumors along with cancer cells, survive in blood circulation and in metastatic nodules. Pre-existence of a tissue-like structure in the form of heterotypic cell fragments may increase the viability of cancer cells in blood circulation and modulate metastasis efficiency. How this mechanism contributes to the organo-tropism of certain metastatic tumors is not known. But demonstration of the involvement of some of the same tumor “soil” in both local and distant tumor invasion has important conceptual and clinical implications for colonization during metastasis progression.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6151.
Implication of a Pro-Apoptotic Protein, Prostate Apoptosis Response-4 (Par-4) in Breast Cancer.
Authors: Ye L. , Mansel R. , Jiang W.
Published: December 2010

Background: Par-4, Prostate Apoptosis Response-4, is pro-apoptotic in certain cell types and has been implicated in the development and progression of solid tumours. However, the role of Par-4 in breast cancer remains unclear. The current study investigated the role played by Par-4 in breast cancer.Methods: The expression of Par-4 was examined in a breast cancer cohort using quantitative real time PCR and immunohistochemistry (IHC). The biological functions of Par-4 in breast cancer cells were tested by creating breast cancer cell lines that had differential Par-4 expression. The cell functions were examined using a variety of in vitro assays, including growth, migration and invasion. The implication of Par-4 in breast cancer was further tested in an in vivo tumour model.Results: A marked decrease of both Par-4 mRNA and protein was seen in breast tumours compared with background tissues. Par-4 staining was cytoplasmic in both mammary epithelial cells and cancer cells in the tissues. The level of Par-4 transcripts in primary tumours was decreased in the patients with poor prognosis and worse clinical outcome. The overall survival was poor in the patients with lower Par-4 transcript level, p=0.0082 compared with the patients who had higher expression. Patients with higher expression level also had longer disease free survival (133.1 months (95% CI, 121.1-145.1 months)), p=0.0597 compared with patients who had lower expression (118.4 months (95% CI, 103.2-133.5 months)). Over-expression of Par-4 in MDA-MB-231 cells (MDA-MB-231Par4exp) resulted a reduction of cell growth, migration and invasion in vitro. The invaded cell number of MDA-MB-231Par4exp was 17.9±2.5, p<0.01 compared with both MDA-MB-231WT(30.8±2.6) and MDA-MB-231pEF(34.1±6.5) controls. This also led to a decreased in vivo tumour growth, in an athymic mice model.Conclusions: Taken together, decreased Par-4 expression in breast cancer correlates with poor prognosis and shorter survival. Par-4 suppresses growth, migration and invasiveness of breast cancer cells in vitro, and also inhibits in vivo tumour growth. It suggests that Par-4 plays critical roles in the progression of breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6155.
Tumor Invasion and Metastasis Initiated by mir-106b in Breast Cancer by Targeting BRMS1 and RB.
Authors: Pan S. , Yu F. , Gong C. , Song E.
Published: December 2010

Background: MicroRNAs (miRNAs) are noncoding RNAs that have recently been demonstrated to have an important role in tumor metastasis. Mir-106b family is reported to promote the proliferation of cancer as an oncogene. However, whether mir-106b has roles in breast cancer metastasis and EMT remains unknown. Therefore, functional characterization of mir-106b in breast cancer cells would potentially facilitate our understanding of the development of breast cancer.Methods: Mir-106b expression and location in primary breast cancer samples from patients with metastasis were compared to the samples without metastasis by in situ hybridization analysis. Differentially expressed miRNAs in MDA-MB-231 breast cancer cell line were quantified by qRT-PCR and Northern Blot followed by scratch assays, migration assay, Boyen chamber assay and invasion assay to determine the significance of mir-106b in metastasis. Lentivirus-mediated miRNA transduction was used to determine the significance of mir-106b function. Target scan and luciferase reporter assay were used to identify the targets of mir-106b. Mir-106b targets' gene expression (BRMS1 and RB) was determined by qRT-PCR and western blot. The expression of E-cadherent by immunofluorescence and Western Blot were used to determine the regulation of mir-106b on the effect of TGF- β induced EMT.Results: Mir-106b was dramatically increased in primary breast cancer samples from patients with metastasis compared to the samples without metastasis, which is negatively correlated with patients' prognosis. MDA-MB-231 cells transfected with lentivirus-mir106b had a higher ability of invasion and migration. Conversely, transfecting mir-106b ASO into MDA-MB-231 cells decreased the invasion and migration ability in vitro. Silencing BRMS1 (breast cancer metastasis suppressor 1) and RB (retinoblastoma), which were proved to be new targets of mir-106b by luciferase assay, showed the similar results with mir-106b over-expressing. Furthermore, the expression of mir-106b in MCF10A cells was increased during TGF-βinduced EMT, while TGF-β-induced EMT was inhibited in MCF10A cells transfected with mir-106b ASO. Furthermore, we found that mir106b-expressing MDA-MB-231 cells gave rise to more tumors and metastasized more frequently in vivo.Conclusion: mir-106b can promote breast cancer invasion and metastasis by targeting BRMS1 and RB. Mir-106b mediates TGF-β-induced EMT, which is an early process of tumor metastasis. Therefore, mir-106b may be an effective maker to predict prognosis of breast cancer patients, and a new targets to treat breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6157.
The Role of Dragon (Repulsive Guidance Molecule-B, RGM-B) in Human Breast Cancer.
Authors: Li J. , Ye L. , Sanders A. , Mansel R. , Jiang W.
Published: December 2010

Introduction: Repulsive guidance molecules (RGMs) are involved in embryonic development and iron homeostasis. RGM-A mediates repulsive axonal guidance and neural tube closure, and RGM-C is mutated in juvenile hemochromatosis. RGM-B, also known as Dragon, is a myelin-derived inhibitor of axon growth in the central nervous system. The RGM family was also identified as co-receptors of bone morphogenetic proteins (BMPs), a group of proteins that are involved in development of bones, the differentiation and progression of cancer. However, the role of RGMs played in breast cancer remains unclear. In the present study, we examined the pattern of expression of the RGM family in human breast cancer cells and investigated the impact of Dragon (RGM-B) on BMP-induced cell function in breast cancer cells.Material and Methods: Conventional RT-PCR was performed to screen the expression of RGMs in human breast cancer samples and a range of breast cancer cell lines. Dragon/RGM-B ribozyme transgenes were generated and in order to knock down the Dragon transcript. Subsequently, MDA-231RGMB-Knock-down variants were created by way of the transgenes. A series of cell function assays were employed to investigate any biological effects upon RGM-B knockdown on the breast cancer cells as well as cell's response to recombinant BMP proteins.Results: RGM-A and RGM-C transcripts were barely detectable in breast cancer cell lines (MDA-MB-231 and MCF-7) and tissues. However RGM-B transcripts were expressed in both cell lines. Using anti-RGM-B transgenes, MDA-231RGMB-Knock-down variant cell lines, in which RGM-B transcripts were knocked down, were created. Compared with wild type and control transfection, MDA-231RGMB-Knock-down variants displayed a significant increase in both the adhesiveness and cells growth (p < 0.05, vs the respective controls, for adheresion and for cell growth). Interestingly, RGM-B knockdown did not have any significant effects on the invasiveness of the cells. Finally, wild type breast cancer cells and the RGMB knock down variant cell lines showed a similar response to the treatment with rhBMP-11, indicating that RGM-B is less critical in BMP-11 mediated cell functions.Conclusion: The present study is the first to examine the role of RGM-B in breast cancer and has demonstrated that knock-down of RGMB could enhance breast cancer cells' ability to grow and attach, indicating that RGM-B may act as an inhibitor in breast cancer. This property is unique to RGMB, as this ability is not associated with any other member in the RGM family.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6158.
Role for CD44 in Enhancing Invasion, Migration, and Growth of Triple Negative (TN) Breast Cancer Cells.
Authors: Iida J. , Nesbella M. , Lehman J. , Mural R. , Shriver C.
Published: December 2010

Triple negative (TN) breast cancers are defined by a lack of expression of estrogen, progesterone, and her-2/neu receptors. It is widely recognized that TN breast cancers have a poorer prognosis than any other subtypes of breast caner. Given the lack of effective targeted therapies for TN breast cancer patients, understanding of the mechanisms of growth and invasion in the tissues provides insight into developing novel approaches to lower the mortality from TN breast cancer.Neoplastic epithelial cells in breast carcinomas interact with various components in the tissue microenvironment including extracellular matrix (ECM) and mesenchymal cells. Recent studies identified CD44 as a metastasis-related molecule with multiple functions by promoting cell-cell and cell-ECM interactions. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. In the standard form (CD44s), 10 of the 20 exons are translated. Multiple variant isoforms exist (CD44v1-10) which arises from alternate mRNA splicing of the remaining 10 exons. In contrast to the ubiquitous expression of the standard form of CD44, splice variants are highly restricted in their expression in normal or malignant tissues. Indeed, CD44 variants containing v3, v5, v6, v7-8, v10 exons are expressed in malignant breast cancer tissues. However, there is limited information regarding the biological functions of these exons to promote tumor invasion and metastasis. The goal of this study is to evaluate specific exon(s) of CD44 expressed on TN breast cancer cells for promoting tumor progression and metastasis.In order to approach this goal, we utilized three TN cell lines (HCC38, HCC1937, and HCC1806) as model systems to evaluate CD44 in regulating invasion, migration, and growth in extracellular matrix (ECM) environments. Reverse transcriptase (RT)-PCR analysis using exon specific primers indicate that these cells expressed CD44v8-v10 and CD44s. We demonstrated that an inhibitory antibody against exon v10 of CD44 significantly inhibited b1 integrin-mediated migration and invasion into Matrigel and type I collagen gel. Importantly, this antibody also inhibited three dimensional (3D) growth which is a b1 integrin-independent process. The significant inhibition of these processes was also achieved when a FLAG-fusion exon v10 peptide (FLAG-v10, in which FLAG is tagged at the N-terminal of the peptide) was used as an inhibitor, implying that this exon would function to assemble molecular complexes on TN breast cancer cells that facilitate invasion, migration, and growth. Thus, these results suggest that generation of small synthetic molecules that block the functions of exon v10 of CD44 is promising approaches to inhibit invasion and metastasis of TN breast cancer cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6161.
Radiation-Induced S-Nitrosylation of ER-α Predisposes MCF-7 Breast Cancer Cells to Aggressive Invasion and Migration.
Authors: Tamfu R. , Natarajan A. , Yu H. , Mohan S. , Shanmugasundaram K. , Natarajan M. , Natarajan M. , Natarajan M.
Published: December 2010

The objective of this study is to decipher the potentials of breast cancer cells that survive radiation exposure at clinical doses to acquire invasive and metastatic determinants. In this study we exposed estrogen receptor positive (MCF-7) and negative (MDAMB-231) breast cancer cells to clinical doses of low LET radiation including doses used as single fraction (2Gy), cumulative dose (10Gy) and scattered dose (10cGy). The cells were exposed to 137Cs source at a dose rate of 1.27 Gy/min. The mock irradiated cells were used as controls. The mRNA transcript level, protein expression and protein-protein interaction were analyzed by QPCR/RT-PCR, immunoblotting, immunoprecipitation and mammalian two-hybrid system, respectively. Intracellular nitric oxide (NO) levels were determined by immunofluorescence and electron spin resonance spectroscopy (ESR) using DAF-FM and Fe-MGD as NO traps, respectively. NO-dependent ER-α s-nitrosylation was determined through immunoprecipitating s-nitrosylated proteins from the cell lysates with an s-nitrosylated-cysteine IgG antibody followed by ER-α western blot analysis. Transactivation of ER-α was measured by EMSA and luciferase reporter assay. Cell invasion and migration were examined by co-culture system using thin inserts.Radiation induces eNOS expression and activation through the phosphorylation of eNOS at Ser1177 site resulting in increased bioavailability of NO in a dose- and time-dependent manner. The three-fold bioavailability of NO s-nitrosylates ER-α leading to a two-fold binding of ER-α to ERE. Paradoxically, this binding did not translate into the transcriptional activation of ER-α dependent gene regulation. Altered structure due to s-nitrosylation of ER-α contributes to enhanced cell invasion and cell migration. In consistent with these results, there was an associated 2.3-fold increase in the expression of MMP-2 and MMP-9 and corresponding TIMP-1 decreased expression leading to a 3.5-fold increase in MMP activity. Radiation, while controlling tumor growth, could simultaneously play a significant role in breast cancer relapse and metastasis through the activation of eNOS and the generation of NO. The bioavailability of NO s-nitrosylates ER-α leading to re-defining the genomic functions of ER-α and thereby impart metastatic transformation potentials to ER-α positive breast cancer cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6162.
The Role of Claudin- 5 and the Paracellular Barrier Function in Endothelial Cells during Invasion of Breast Cancer Cells.
Authors: Escudero-Esparza A. , Martin T. , Jiang W.
Published: December 2010

Material and Methods: Human endothelial (HECV) and breast cancer cells (MDA-MB-231) were used to assess the effect of over-expression (HECVCL-5, MDACL-5) or knockdown (HECVDCL-5, MDADCL-5) of Claudin-5. Cell adhesion and growth were evaluated using in vitro assays. An in vivo tumour model using athymic nude mice was used to assess growth of tumours. Transendothelial and transepithelial resistance (TER) was employed to measure changes in TJ function. Cellular motility was analyzed using Cytodex-2 bead motility assay. ECIS (electrical cell impedance sensing) was used to assess the attachment and migration of the cells.Results: There were no significant differences in in vitro growth of cells that over-expressed or had knockdown of Claudin-5 (both HECV and MDA-MB-231 cells). Expression of CL5 in both HECV and MDA-MB-231 did not have a significant impact on in vivo growth. There were however, significant differences in adhesion between control cells (HEVCpEF6) and HECVCL-5, HECVDCL-5 (changes in adhesion: HEVC pEF6 vs. HECVCL-, P>0.05; HECVDCL-5, P=0.046). There was a distinct modulation of TJ function as assessed using TER. HECVDCL-5 cells exhibited increased resistance compared to HEVC pEF6 and HECVCL-5 (change in TER: HEVC pEF6: -151.33±6.11 vs. HECVCL-5 -187±1.73, HECVDCL-5-89.33±2.08, P <0.01). There was no difference in TER in MDA CL-5 and MDADCL-5cells. HGF, a powerful motogen and modulator of TJ function, caused a significant increase in TER in HECVCL-5 and HECVDCL-5, but resulted in a significant decrease in TER in MDA-MB-231 cells. Motility studies revealed that HECVCL-5 and MDA CL-5 after treatment with HGF cells were significantly more motile than cells without treatment (HECVCL-5 vs. HECVCL-5+HGF, P=0.015; MDACL-5 vs. MDACL-5+HGF, P=0.033). There was no difference in motility in HECVDCL-5 cells, however, MDADCL-5 cells were significantly less motile after treatment with HGF (P=0.017). Interestingly, ECIS results showed that the ROCK inhibitor Y27632 inhibited the attachment and migration of HECVCL-5 cells but was no longer able to inhibit attachment and migration of HECVDCL-5 cells.Discussion: These results demonstrate for the first time that Claudin-5 can be modulated to effect changes in cell motility, attachment and TJ function in human endothelial and breast cancer cells. Moreover, there appears to be a link between Claudin-5 expression and cell motility, particularly with respect to the ROCK signalling cascade.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6170.
The Role of Securin in Tumorigenesis and Metastasis.
Authors: Hatcher R. , Zhang P.
Published: December 2010

Genomic instability is a hallmark of human cancers. Spindle assembly checkpoint (SAC) is a critical cellular mechanism that prevents chromosome missegregation and aneuploidy by blocking premature separation of sister chromatids. The process of sister-chromatid separation occurs when separase cleaves Scc1 of the cohesin complex holding sister-chromatids together. The cell prevents this event from taking place untimely by establishing regulatory mechanisms on separase. Thus, the SAC, much like the DNA damage checkpoint, is essential for genome stability.Although a large body of evidence supports an intimate relationship between defects in the DNA damage checkpoint and tumorigenesis, such evidence is lacking for the spindle assembly checkpoint. Attempts to disable SAC by inactivating individual component, such as Mad2, or BubR1, yielded only unviable mice.Securin was the first identified inhibitor of separase in budding yeast as PDS1 (1, 2). Deletion of PDS1 caused genome instability and lethality when treated with nocodazole (2). Based on these results, it was hypothesized that deletion of the securin gene would accelerate tumorigenesis in mammals through inducing aneuploidy.However, when securin was knocked out in mice, the mutant mice were essentially normal and the cells derived from these mice show an intact SAC (3, 4). Although, securin null cells are compromised in their ability to grow in the presence of sub-lethal doses of nocodazole (4), indicating that securin does contribute to the proper functioning of the checkpoint. The over-expression of securin has been associated with metastasis in human breast cancer and therefore my securin knockout mice have not developed tumors.Apparently securin gain-of-function or loss-of-function does not have strong enough oncogenic activity to initiate tumor development, but perhaps in combination with a stronger oncogene tumors will develop faster and more aggressively. To test this, I am combining securin knockout with MMTV-ErbB2 transgenic mice, which develop adenocarcinomas in the mammary tissue by approximately 6 months old (5).Given the association of securin expression with metastasis, it is possible that securin may have a role in tumor cell metastasis. To test that possibility, I am taking advantage of the MMTV-ErbB2 mice again as these mice show lung metastasis by approximately 8 months old (5). Therefore, my hypothesis is that loss of securin facilitates tumor formation by destabilizing the genome as a result of mitotic errors, but may hinder metastasis because of its cell cycle unrelated functions.References:1. Yamamoto, A., Guacci, V., and Koshland, D. (1996a). J. Cell Biol. 133, 85–97.2. Yamamoto, A., Guacci, V., and Koshland, D. (1996b). J. Cell Biol. 133, 99–110.3. Mei, J., Huang, X., and Zhang, P. (2001). Curr. Biol. 11, 1197–1201.4. Huang, X., Hatcher, R., York, P., Zhang, P.(2005). Mol. Bio. of the Cell. 16, 4725–4732.5. Muller WJ; Sinn E; Pattengale PK; Wallace R; Leder P. 1988. Cell 54(1):105-15.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6172.